Antimicrobial activity of lactic acid bacteria from sour milk products "Narine", "Karine", and "Matsun"

We studied antimicrobial properties of lactic acid bacteria from sour milk products Narine, Karine, and Matsun. The whey of sour milk products included two major fractions of sugars and L-lactic acid and its sodium and calcium salts. Antimicrobial activity of Narine, Karine, and Matsun was related to the presence of L-lactic acid and its sodium and calcium salts.     

Effect of various salts on the growth and development of Geotrichum candidum,the causal agent of carrot sour rot

This study evaluated the efficacy of ammonium, calcium, potassium and sodium salts as possible alternatives to synthetic fungicides in the control of Geotrichum candidum, the causal agent of sour rot on carrots. In vitro mycelial growth of G. candidum was completely halted by ammonium bicarbonate and carbonate; calcium oxide; potassium benzoate, carbonate and sorbate; sodium benzoate, carbonate and fluoride (2% w/v). Potassium and sodium bicarbonate also reduced mycelial growth by 77.78% and 90.60%, respectively, and the difference between the effects of sodium bicarbonate and the first group of salts was not statistically significant (p < 0.05). With the exception of potassium and sodium bicarbonate, the above‐mentioned salts also halted or strongly reduced arthrospore germination. Potassium bicarbonate, and sodium bicarbonate, acetate and propionate significantly increased conidiation (p < 0.05). Of all the salts tested in vitro, only ammonium bicarbonate and carbonate, calcium oxide and sodium fluoride were toxic to G. candidum. In in vivo studies, all the calcium salts tested (acetate, chloride, citrate, formate, lactate, oxide, propionate and silicate), several of the sodium salts (acetate, bicarbonate, chloride and fluoride) and potassium bicarbonate exhibited both protective and curative activity against G. candidum, significantly reducing the severity of sour rot in comparison to pathogen‐inoculated controls (p < 0.05). Although no curative was observed with ammonium bicarbonate, ammonium carbonate, potassium carbonate, potassium chloride, sodium carbonate or sodium citrate, these salts also demonstrated significant protective activity against sour rot when compared to controls (p < 0.05). In sum, the study findings show that all of the selected salts may be used to control carrot sour rot, except for sodium fluoride, which exhibited phytotoxicity to carrots.     

Rheological properties and volatile composition of fermented milk prepared by exopolysaccharide-producing <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> n.v. Er2 317/402 strain Narine

Fermented milk products were made using a probiotic strain of human origin, Lactobacillus acidophilus n.v. Er2 317/402 strain Narine. The effects of the strain on the texture, rheology, microstructure, and volatile profiling of fermented milk products over a period of 25 days under refrigerated storage conditions were investigated using steady and dynamic rheological tests, confocal laser scanning microscopy (CLSM), and headspace solid-phase microextraction gas chromatography mass spectrometry, respectively. CLSM images confirmed that exopolysaccharides (EPSs) from Narine were large in size and interacted with proteins. EPSs (50 mg) were obtained after 8 h of incubation. Volatome profiling of Narine fermented milk revealed 15 volatile compounds, including 2-heptanone, 2-nonanone, acetoin, isobutyric acid, isovaleric acid, and 2-phenylethanol. EPSs produced from L. acidophilus n.v. Er2 317/402 strain Narine gave the fermented milk a smooth ropy texture. The natural, soft-bodied, drinkable, and pleasant flavor of Narine makes it an attractive food ingredient.     

Genetic improvement of technological characteristics of starters for fermented milk products

Possibility for improvement of technological characteristics of lactobacilli using mutations of resistance to rifampicin (rif ^r ) and streptomycin (str ^r ) was studied. Using starter model of Narine Lactobacillus acidophilus INMIA-9602 Armenian diet milk product, it was showed that a possibility for selecting strains with increased rate of milk fermentation and acid production is higher in Rif^r and Str^r mutants induced by nitrosoguanidine than in cultures sensitive to antibiotics. The milk products obtained using Rif^r and Str^r strains had high viscosity, improved texture, increased amount of alive cells and good organoleptic features.     

Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.     

Membrane-Integrated Fermentation System for Improving the Optical Purity of D-Lactic Acid Produced during Continuous Fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Reducing by-product formation in L-lactic acid fermentation by Rhizopus oryzae

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l^–1 to 0.6 g l^–1 KH₂PO₄ reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l^–1 to 71 g l^–1 and from 1.36 g l^–1 to 0.18 g l^–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l^–1 KH₂PO₄ was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.     

Direct fermentation of corn to L(+)-lactic acid byRhizopus oryzae

Summary Rhizopus oryzae NRRL 395 was found capable of fermenting ground corn directly to L(+)-lactic acid in the presence of calcium carbonate. The average yield of L(+)-lactic acid was more than 44% on the basis of the amount of total carbohydrate as glucose consumed.     

Production of algal gels from the brown alga, <Emphasis Type="Italic">Laminaria japonica</Emphasis> Aresch., and their biotechnological applications

Alginic acid is localised in the cell walls and intercellular spaces of the brown alga, Laminaria japonica Aresch., and the salts of this compound occur mainly as calcium alginates. However, the alginates in this alga do not have the viscosity and the ability to create and stabilise structural products. Hence, the structure and properties of the alginates in Laminaria were changed by chemical modification to produce new products such as sodium alginates and other substances capable of forming gels. The rheological properties of the algal gel from Laminaria depended on the properties of the sodium alginate. Heating the algal product up to 90°C did not change its physical and chemical properties; the viscosity did not differ from that of the initial product. The viscosity and molecular weight of the sodium alginate isolated from the algal gel were stable from 20°C up to 95°C. However, about 30% of the viscosity was lost at 100°C. Recipes and various methods of preparing the gel products as fish sauces, jelly-like fish products, fruit jellies, drinks, cosmetic and pharmaceutical products are presented. The algal gel and the gel products did not lose their integrity with heat processing. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Use of organic acids and salts to control postharvest diseases of lemon fruits in Egypt

Abstract

Postharvest diseases caused by Geotricum candidum (sour rot), Penicillium digitatum (green mould), and P. italicum (blue mould) are the most important negative factors affecting handling and marketing of citrus fruits in Egypt. The effect of organic acids (ascorbic, benzoic, citric and sorbic) as well as organic salts (potassium sorbate and sodium benzoate) were evaluated on the growth of causal agents and their disease incidence under in vitro and in vivo conditions. Complete inhibition was observed in the linear growth of all tested fungi when exposed to benzoic, citric and sorbic organic acids at concentrations of 4% and 2% of either sodium benzoate or potassium sorbate, respectively. Minimizing the tested concentration of organic acid down to 2%, the tested fungi fluctuated in their response such that only benzoic and sorbic acids could completely inhibit the growth of either P. digitatum or P. italicum only. Different organic acids and salts showed various levels of either protective or therapeutic effect for coated lemon fruits against mould infection whatever the time of their artificial inoculation under in vivo conditions. All treated fruits showed reduction in sour rot and green and blue mould diseases when compared with untreated fruits. Complete inhibition of mould incidence was obtained in coated lemon fruits with 4% of water or wax mixtures of sodium benzoate and potassium sorbate 24 hours before inoculation. Also, high reduction in mould incidence was observed in lemon fruits coated with the same concentration at 48 hours after inoculation under the same conditions. On the other hand, the tested organic acids showed a lesser effect on mould incidence. Moreover, they were more effective against mould incidence when dissolved in water than wax, that only 4% of water mixture of sorbic and benzoic acids showed 100% protection against mould incidence. Furthermore, the severity of infection records followed the same trend. The present findings demonstrate that potassium sorbate and sodium benzoate have potential as environmentally friendly products, nontoxic postharvest fungicides against sour rot, green and blue mould incidence of stored citrus fruits and could be suggested for commercial use in packing-houses in consideration to their wide consumption as safely food preservatives.     

Untersuchungen zur Entstehung von Dl-Milchsäure bei Lactobacillen und Charakterisierung einer Milchsäureracemase bei einigen Arten der Untergattung Streptobacterium

Zusammenfassung Eine kritische Überprüfung der von den verschiedenen Arten der Gattung Lactobacillus gebildeten Milchsäure ergab, daß zwar die D(-)-Laktatbildner reines D(-)-Isomer, die L(+)-Laktatbildner aber immer auch einige wenige Prozente des anderen Isomers bilden. Letzteres beruht auf der Anwesenheit einer sehr schwach aktiven NAD-abhängigen D-Laktatdehydrogenase neben der hochaktiven NAD-abhängigen L-Laktatdehydrogenase.Die Bildung von Dl-Laktat beruht entweder auf der Anwesenheit der beiden stereospezifisch verschiedenen Laktatdehydrogenasen oder auf der Bildung vo L(+)-Milchsäure und anschließender Racemisierung. In einigen Fällen ist die biochemische Grundlage der Racematbildung noch unklar. Bei fast allen Dl-Bildnern ist das Isomerenverhältnis von den Wachstumsbedingungen abhängig.Bei Lactobacillus curvatus, L. sake und L. casei ssp. pseudoplantarum wurde die für die Dl-Bildung verantwortliche Milchsäureracemase nachgewiesen und näher charakterisiert. Es handelt sich um ein induzierbares Enzym, dessen Induktor L(+)-Milchsäure ist. Die Induktion kann mit Actinomycin D gehemmt werden. Eine Trennung von Racemase und L-Laktatdehydrogenase gelang durch Zentrifugation im Saccharosedichtegradienten, da das Molekulargewicht der Racemase mit 52–60000 in einigen Fällen niedriger liegt als das der Laktatdehydrogenase.

---

Formation of Dl-lactic acid by lactobacilli and characterization of a lactic acid racemase from several streptobacteria

Summary The isomer composition of the lactic acid formed by the various species of lactobacilli was enzymatically determined. It is shown, that the D(-)-lactate formers produce D(-)-lactate exclusively whereas all L(+)-lactate formers always produce a few per cent of the other isomer in addition. The latter is due to the presence of a NAD dependent D-LDH of very low activity. The formation of Dl-lactic acid is either caused by the presence of both stereospecific different LDHs or by the formation of L(+)-lactic acid followed by racemisation. In some species the biochemical basis of the racemate formation is still unknown.The racemase was isolated and characterised from Lactobacillus curvatus, L. sake and L. casei ssp. pseudoplantarum. This enzyme is induced by L(+)-lactic acid. The induction is inhibited by actinomycine D. A separation of lactic acid racemase and of L-LDH was achieved by the application of a sucrose density gradient.

     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.     

Elucidation of the Taxonomic Status of Industrial Strains of Thermophilic Lactic Acid Bacteria by Sequencing of 16S rRNA Genes

Both phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 520–525.Original Russian Text Copyright © 2005 by Botina, Lysenko, Sukhodolets.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Metabolic engineering of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> for the production of platform chemicals

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.     

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264. Received 05 November 2000/ Accepted in revised form 03 July 2001     

Formation of aminium lactates in lactic acid fermentation preparation and characterization of 1,4-piperazinium-(L,L)-dilactate obtained from L(+)-lactic acid (part I)

The potential for the production of 1,4-piperazinium-(L, L)-dilactate from L(+)-lactic acid preparations obtained by fermentation was studied. Piperazinium dilactate was found to be a very suitable source material for poly(lactic acid) production. In a novel polymerization process, the intermediate dilactide was directly formed in the salt melt at a moderate temperature. High-performance cultivation of Lactobacillus paracasei on a glucose-MRS medium was carried out using high-viability inocula. After the cell mass had been removed from the fermentation broth by centrifugation and/or ultrafiltration, the lactic acid solution was concentrated to 45% [w/w] by a two-stage electrodialysis process. Two methods of preparing 1,4-piperazinium dilactate were developed: the first from the medium-concentrated lactic acid (45%) and the second from a highly-concentrated lactic acid (85%) obtained by evaporation from the first one. Because there were no physical data on 1,4-piperazinium-(L, L)-dilactate in specialized literature, the pure product was characterized according to its solubility characteristics, melting point and spectroscopic analysis.     

Anti-hypertensive activity of fermented dairy products containing biogenic peptides

A considerable amount of research has been conducted to study the health benefits of fermented milks. One possible mechanism for the beneficial effects is the involvement of biogenic compounds, which are produced by the action of microorganisms and make the products directly beneficial without the need for live bacteria. Research on the health effects of a pasteurized sour milk, which is fermented by a starter culture containing Lactobacillus helveticus as the predominant microorganism, indicated clear evidence for the involvement of biogenic compounds. In this paper, beneficial effects of sour milk are reviewed with special attention to the effect on anti-hypertension and angiotensin-I converting enzyme inhibitory peptides identified in sour milk.     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad