Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly than _s-cascin.     

Contribution of Lactococcus lactis subsp lactis IFPL 359 and Lactobacillus casei subsp casei IFPL 731 to the Proteolysis of Caprine Curd Slurries

ABSTRACT: Proteolysis was studied in low-fat caprine curd slurries pre-incubated (24 h at 30 °C) with an extra dose of rennet, 10.0 or 22.0 μg g^-1 (LR and HR slurries, respectively), and treated at 70 °C for 15 min prior to incubation with sonicated cells of Lactococcus lactis subsp lactis IFPL 359 alone or combined with Lactobacillus casei subsp casei IFPL 731. The addition of lactobacilli to slurries containing lactococci increased the conversion of water-soluble nitrogen to nonprotein nitrogen and amine nitrogen. Major differences were found for some free amino acids, such as glutamic acid and proline, and hydrolysis of most hydrophobic peptides produced by lactococci, which are associated with bitterness in cheese.     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

The accelerating effect of aLactococcus lactis subsp.lactis lactose-utilization/proteinase-deficient strain on proteolysis and flavour development

This study describes the accelerating effect exerted on proteolysis and flavour development in cheese-curd slurries by combined application ofLactococcus lactis subsp.lactis IFPL359 and high concentrations of its Lac^? Prt^? derivative, (i.e. that which has reduced capacity to metabolize lactose and reduced proteolytic activity, strain T1). Cells of strain T1 partially lysed by either sonication or incubation with lysozyme, were also used to ascertain how proteolysis was affected by release of intracellular enzymes in the initial stage of incubation of the strains in cheese-curd slurries. The presence of strain T1 produced higher levels of non-protein nitrogen (NPN) and amine nitrogen (AN) during the first weeks of incubation when partially lysed cells had been added. Addition of whole cells of strain T1 produced higher values of NPN and AN at the end of incubation of slurries, accelerating proteolysis by about 2 weeks with respect to the control, which only contained the parental strain IFPL359. At the end of the experimental period higher amino acid levels were detected by HPLC in the slurry containing whole T1 cells. Volatile fractions of the different cheese-curd slurries were also analysed. The higher level of proteolysis produced by addition of high levels of strain T1 appeared to be related to release of intracellular enzymes by this strain owing to its greater capacity for autolysis.     

The physicochemical surface characteristics of Lactobacillus casei

The surface and interfacial properties of five strains of Lactobacillus casei, commonly found in cheese, were investigated in an attempt to identify surface properties that might be exploited to effect adsorption of the cells onto milk fat globules. All measures of surface hydrophobicity revealed these strains to be moderately hydrophobic compared to a reference strain of Mycobacterium smegmatis which was very hydrophobic. In chromatography experiments, L. casei had no affinity for either hydrophobic (octyl-sepharose) or control (sepharose) columns in conditions of physiological pH and low ionic strength, despite a favorable free energy of interfacial interaction (ΔG^IF_IW2) as derived from contact angle measurements. Adhesion of L. casei strains to these materials was effected by manipulation of eluent pH and ionic strength. In contrast, adhesion of L. casei strains to n-hexadecane concurred with ΔG^IF_IW2. Surfaces of L. casei strains subjected to X-ray photoelectron spectroscopy exhibited similar amounts of carbon and oxygen but displayed considerable diversity in surface nitrogen and phosphorous contents. Carbon existed predominantly as C-(O, N) and oxygen as -OH on L. casei surfaces but carbon on M. smegmatis surfaces was present predominantly as C-(C,H).     

Extraction and partial characterization of proteolytic activities from the cell surface of Lactobacillus helveticus Zuc2

Proteolytic activities were extracted from a dairy Lactobacillus helveticus strain and partially characterized. A first cell envelope proteinase (CEP) was extracted using a high ionic strength buffer, both in the presence and in the absence of Ca²⁺. Moreover, cell treatment by 5 M LiCl allowed for the selective removal of the S-layer protein and CEP, suggesting an enzyme ionic linkage to the cell envelope similar to that observed for the Slayer structure. The enzyme specificity against α_s1-CN (f1-23) showed unusual activity on the Lys₃-His₄ bond compared with other proteinases of the same species. A second proteinase appeared to be linked to the cell membrane because it was extractable only after membrane disgregation by detergents. Its specificity against CN fractions and α_s1-CN (f1-23) was different from that of the first CEP; moreover, the measured activity was lower than that of CEP.     

Control of late blowing in cheese by adding lacticin 3147-producing Lactococcus lactis IFPL 3593 to the starter

Bacteriocins produced by lactic acid bacteria offer potential as tools for ensuring food safety and quality. Lacticin 3147 is a two-peptide lantibiotic previously shown to inhibit a broad range of food spoilage and pathogenic bacteria. Lacticin 3147-producing Lactococcus lactis IFPL 3593 was investigated for its ability to inhibit the growth of clostridia as a means to prevent late blowing in cheese. L. lactis IFPL 3593 was shown to inhibit germination of clostridia spores and prevent late blowing in semi-hard cheeses, with a 5 log g^?1 reduction in the numbers of spores when compared to control cheeses. Furthermore, this bacteriocin-producing strain demonstrated considerable potential as a biopreservative agent against heterofermentative lactobacilli and their associated blowing defects. The use of this strain to prevent late blowing in cheese thereby represents a promising alternative to the addition of lysozyme particularly given the increasing concerns regarding the potential allergenicity of this additive.     

Expression of amino acid converting enzymes and production of volatile compounds by Lactococcus lactis IFPL953

Lactococcus lactis contains a large number of key enzymes responsible for the formation of volatile compounds characteristic of cheese flavour. In the present work we have investigated the expression of genes codifying amino acid converting enzymes (AACE) and the formation of volatile compounds by L. lactis IFPL953 and its mutant lacking the hydroxy acid dehydrogenase PanE (L. lactis IFPL953ΔpanE). The growth in absence of isoleucine was the main induction factor in the expression of araT, bcaT, panE, and kivD. The expression of the gdh gene of L. lactis IFPL953ΔpanE increased remarkably during the stationary growth phase, particularly under isoleucine and valine starvation conditions. L. lactis IFPL953ΔpanE showed an enhanced formation of 2- and 3-methylbutanal and their corresponding alcohols and an increase in the formation of ketones. These findings contribute to a better knowledge of L. lactis AACE regulation and its potential application in cheese flavour formation.     

Purification and characterization of CEP from Lactococcus lactis ssp. lactis

The cell-envelope proteinase (CEP) of Lactococcus lactis ssp. lactis LB12 was released from cells by treatment with lysozyme, purified by ammonium sulfate precipitation and chromatographed on DEAE-Sephadex A-25 and Sephacryl S-300 HR. The purified CEP is a monomer structure and has molecular mass of about 53 kDa. Optimal activity occurred at pH 7.5 and 40 °C. It is a metallopeptidase, activated by Mn²⁺, Mg²⁺, Ca²⁺, inhibited by Co²⁺, Zn²⁺, Ni²⁺ and EDTA, and a serine proteinase which is inhibited by PMSF. The sequence of the first 13 amino acids of the N-terminal of the CEP was determined to be Asp-Val-Phe-Ala-Pro-His-Met-Ala-Asn-Val-Ala-Ala-Val, and the whey protein hydrolysate produced by the CEP displayed ACE-inhibitory activity.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Formation of methional by Lactococcus lactis IFPL730 under cheese model conditions

The present work describes the capacity of Lactococcus lactis to produce methional and other sulphur compounds derived from methionine (Met) in a cheese model system. Cheese slurries were prepared from pasteurized ewes' skimmed milk, chemically acidified with glucono-'-lactone and homogenized aseptically adding Met, !-ketoglutarate, pyridoxal 5'-phosphate, thiamine pyrophosphate and NaCl. Slurries were incubated at 12 °C for 14 days with cellular suspensions of L. lactis IFPL359, L. lactis IFPL730 and L. lactis NCDO763 in different combinations. Slurries added with resting cells and the intracellular fraction from L. lactis IFPL730 showed the highest production of methional at the outset of incubation, which decreased during incubation along with a concomitant increase in 3-methylthiopropanol. The sensorial analysis of slurries indicated a characteristic methional aroma (cooked potato-like) in samples containing 4-methylthio-2-ketobutyrate and the intracellular fraction from L. lactis IFPL730. As incubation proceeded, the intensity of methional aroma decreased but samples were judged by the panel tasters as developing a cheese-like flavour.     

The accelerating effect of aLactococcus lactis subsp.lactis lactose-utilization/proteinase-deficient strain on proteolysis and flavour development

This study describes the accelerating effect exerted on proteolysis and flavour development in cheese-curd slurries by combined application ofLactococcus lactis subsp.lactis IFPL359 and high concentrations of its Lac^– Prt^– derivative, (i.e. that which has reduced capacity to metabolize lactose and reduced proteolytic activity, strain T1). Cells of strain T1 partially lysed by either sonication or incubation with lysozyme, were also used to ascertain how proteolysis was affected by release of intracellular enzymes in the initial stage of incubation of the strains in cheese-curd slurries. The presence of strain T1 produced higher levels of non-protein nitrogen (NPN) and amine nitrogen (AN) during the first weeks of incubation when partially lysed cells had been added. Addition of whole cells of strain T1 produced higher values of NPN and AN at the end of incubation of slurries, accelerating proteolysis by about 2 weeks with respect to the control, which only contained the parental strain IFPL359. At the end of the experimental period higher amino acid levels were detected by HPLC in the slurry containing whole T1 cells. Volatile fractions of the different cheese-curd slurries were also analysed. The higher level of proteolysis produced by addition of high levels of strain T1 appeared to be related to release of intracellular enzymes by this strain owing to its greater capacity for autolysis.     

Effect of alsD deletion and overexpression of nox and alsS on diacetyl and acetoin production by Lacticaseibacillus casei during milk fermentation

Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.     

The biological activity of fermented milk produced by Lactobacillus casei ATCC 393 during cold storage

Lactobacillus casei ATCC 393 is an important probiotic strain widely known in dairy technology. However, its capability to produce bioactive peptides from milk proteins has not been studied. The viability of the Lb. casei ATCC 393 strain and some physicochemical properties in fermented milk throughout storage for 21 days at 4 °C was evaluated; biological activity, i.e., antioxidant, angiotensin converting enzyme inhibitory and anticancer activities of water soluble extract and its filtrate (< 2 kDa; F1) were determined. Lb. casei counts remained over 9 log cfu g⁻¹ during the storage period in fermented milk. These bioactivities were increased significantly (P < 0.01) during storage. F1 of fermented milk after three weeks of storage showed the highest bioactivity impact. De novo sequencing assay for peptide identification was applied to the mass spectrum of F1. The promising capability of Lb. casei ATCC 393 to release bioactive peptides from milk proteins was demonstrated.     

Functional analysis of the second methyltransferase in the bacteriophage exclusion system of Lactobacillus casei Zhang

The antiphage ability is an important feature of fermentation strains in the dairy industry. Our previous work described the bacteriophage exclusion (BREX) system in the probiotic strain, Lactobacillus casei Zhang. The function of L. casei Zhang pglX gene in mediating 5′-ACRC^m6AG-3′ methylation was also confirmed. This study aimed to further dissect the function of the BREX system of L. casei Zhang by inactivating its second methyltransferase gene (LCAZH_2054). The methylome of the mutant, L. casei Zhang Δ2054, was profiled by single-molecule real-time sequencing. Then, the cell morphology, growth, plasmid transformation efficiency, and stability of the wildtype and mutant were compared. The mutant did not have an observable effect in microscopic and colony morphology, but it reached a higher cell density after entering the exponential phase without obvious increase in the cell viability. The mutant had fewer 5′-ACRC^m6AG-3′ methylation compared with the wildtype (1835 versus 1906). Interestingly, no significant difference was observed in the transformation efficiency between the 2 strains when plasmids without cognate recognition sequence (pSec:Leiss:Nuc and pG⁺host9) were transformed, contrasting to transforming cells with cognate recognition sequence-containing plasmids (pMSP3535 and pTRKH2). The efficiency of transforming pMSP3535 into the LCAZH_2054 mutant was significantly lower than the wildtype, whereas an opposite trend was seen in pTRKH2 transformation. Moreover, compared with the wildtype, the mutant strain had higher capacity in retaining pMSP3535 and lower capacity in retaining pTRKH2, suggesting an unequal tolerance level to different foreign DNA. In conclusion, LCAZH_2054 was not directly responsible for 5′-ACRC^m6AG-3′ methylation in L. casei Zhang, but it might help regulate the function and specificity of the BREX system.     

Discrimination of dairy industry isolates of the Lactobacillus casei group

Lactobacilli are a major part of the microflora of the gut and of many fermented dairy products, and are found in a variety of environments. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus zeae form a closely related taxonomic group within the facultatively heterofermentative lactobacilli. The classification and nomenclature of these bacteria are controversial. In this study, relationships between these species were investigated using type strains and dairy industry isolates examined with DNA-based techniques and conventional carbohydrate use tests. Carbohydrate use patterns gave poor discrimination of some species, but DNA PCR using specific primers targeted to sequences of the 16S rRNA gene discriminated 4 types consistent with the currently recognized species. Pulsed-field agarose gel electrophoresis of chromosomal NotI restriction fragments identified 18 different band patterns from 21 independent Lactobacillus isolates and confirmed the identity of L. casei strains from 2 culture collections (CSCC 5203 and ASCC 290), both representing the type strain of L. casei. Some isolates were reclassified as L. rhamnosus, suggesting that the prevalence of L. rhamnosus as a natural component of the microflora of dairy foods and dairy environments has previously been underestimated. These methods can provide a practical basis for discrimination of the species and identification of individual industrial strains.     

Stimulatory effects of tea supplements on the propagation of Lactobacillus casei in milk

The influence of several Chinese teas on the ability of Lactobacillus casei to ferment milk was assessed. The three kinds of tea significantly stimulated the growth and acidification of L. casei. The viable cell counts in tea-supplemented fermented milk were higher than those of the control and were maintained during cold storage. Flavour component content increased significantly, and an additional eight components were detected in fermented milk supplemented with 5% green tea infusion (GTI_5%) compared with control fermented milk. Moreover, the increase in total free amino acids after fermentation in GTI_5% was 2.5-fold higher than in the control, suggesting that tea stimulated the growth and metabolism of L. casei during milk fermentation. Higher cell numbers, reduced fermentation time, abundant flavour components and free amino acids were achieved during the tea-supplemented fermentation of L. casei. Green tea effectively enhances the growth of L. casei, therefore could be considered for industrial purposes.     

Purification and characterization of an endopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The K_M value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co²⁺ and Mg²⁺, no effect was observed with Ca²⁺. After inhibition with EDTA, enzyme activity could be restored fully by Co²⁺. Activity was inhibited by Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. The N-terminal sequence of the endopeptidase was determined as: H₂N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H.     

Characterization of the angiotensin-converting enzyme inhibitory activity of fermented milks produced with Lactobacillus casei

Our study assayed angiotensin-converting enzyme (ACE) inhibitory activity and fermentation characteristics of 41 food-originated Lactobacillus casei strains in fermented milk production. Twenty-two of the tested strains produced fermented milks with a high ACE inhibitory activity of over 60%. Two strains (IMAU10408 and IMAU20411) expressing the highest ACE inhibitory activity were selected for further characterization. The heat stability (pasteurization at 63°C for 30 min, 75°C for 25 s, and 85°C for 20 s) and resistance to gastrointestinal proteases (pepsin, trypsinase, and sequential pepsin/trypsinase treatments) of the ACE inhibitory activity in the fermented milks produced with IMAU10408 and IMAU20411 were determined. Interestingly, such activity increased significantly after the heat or protease treatment. Because of the shorter milk coagulation time of L. casei IMAU20411 (vs. IMAU10408), it was selected for optimization experiments for ACE inhibitory activity production. Our results show that fermentation temperature of 37°C, inoculum density of 1 × 10⁶ cfu/g, and fermentation time of 12 h were optimal for maximizing ACE inhibitory activity. Finally, the metabolite profiles of L. casei IMAU20411 after 2 and 42 h of milk fermentation were analyzed by ultra-HPLC electron spray ionization coupled with time-of-flight mass spectrometry. Nine differential abundant metabolites were identified, and 2 of them showed a strong and positive correlation with fermented milk ACE inhibitory activity. To conclude, we have identified a novel ACE inhibitory L. casei strain, which has potential for use as a probiotic in fermented milk production.     

Effects of nitrates and nitrites on Lactobacillus helveticus and Lactobacillus casei dairy cultures

 The aim of this work was to observe the effects of nitrites and nitrates on the titratable acidity of milk after addition of Lactobacillus helveticus (TX 121) and L. casei (CAD 154) dairy cultures and on the levels of nitrate and nitrite. After adding L. helveticus and nitrites the increasing concentrations of the latter brought about a marked decrease in titratable acidity. In milk containing 100 mg·kg^–1 nitrite the resulting concentration was 39.33 mg·kg^–1 NaNO₂. Nitrates caused a less obvious decrease in titratable acidity, giving 13.27 g·l^–1 lactic acid. In milk containing 100 mg·kg^–1 NaNO₃ the resulting concentration was 24.99 mg·kg^–1 NaNO₃. Experiments with L. casei and a nitrite additive revealed a decrease in titratable acidity to 8.89 g·l^–1 lactic acid. After incubation, nitrite levels were reduced from 100 mg·kg^–1 NaNO₂ to 37.81 mg·kg^–1 NaNO₂. Nitrates were also stated to inhibit the titratable acidity of the sample, which decreased to 11.42 g·l^–1 lactic acid. Nitrates were reduced to 46.99 mg·kg^–1 NaNO₃. The present study shows that nitrites, more than nitrates decrease the titratable acidity of milk after addition of L. helveticus and L. casei dairy cultures. A reduction of nitrates and nitrites in milk samples by Lactobacillus was also found. The results of this study can be used in the dairy industry in the production of several types of hard cheese as well as fermented milk products that use L. helveticus and L. casei dairy cultures. Received: 31 March 1999 / Revised version: 6 September 1999