Ethanol from lactose in salted cheese whey by recombinant Saccharomyces cerevisiae

Seven yeast recombinants were selected from 57 fusant colonies on the basis of higher DNA content, nuclear diameter and ethanol yield compared to parental Saccharomyces cerevisiae ATCC 4126 and Kluyveromyces lactis CBS 683. Six recombinants out of the above colonies revealed growth on lactose and sucrose, indicating that they are S. cerevisiae with transformed β-galactosidase systems. The fusant colonies were investigated with respect to their capacity to convert lactose in salted cheese whey into ethanol. Among these recombinants that showed high tolerance towards sodium chloride and higher ethanol yield than lactose fermenting parental K. lactis CBS 683, SK-1 exhibited high tolerance up to 4 g dl^–1 sodium chlonride with an ethanol yield of 4.66 ml dl^–1 (v/v) , SK-23 tolerated 6 g dl^–1 sodium chloride with an ethanol yield of 4.14 ml dl^–1 (v/v) and SK-26 showed resistance towards 8 g dl^–1 sodium chloride and give an ethanol yield of 3 ml dl^–1 (v/v).     

Ethanol from lactose in salted cheese whey by recombinant Saccharomyces cerevisiae

 Seven yeast recombinants were selected from 57 fusant colonies on the basis of higher DNA content, nuclear diameter and ethanol yield compared to parental Saccharomyces cerevisiae ATCC 4126 and Kluyveromyces lactis CBS 683. Six recombinants out of the above colonies revealed growth on lactose and sucrose, indicating that they are S. cerevisiae with transformed β-galactosidase systems. The fusant colonies were investigated with respect to their capacity to convert lactose in salted cheese whey into ethanol. Among these recombinants that showed high tolerance towards sodium chloride and higher ethanol yield than lactose fermenting parental K. lactis CBS 683, SK-1 exhibited high tolerance up to 4 g dl^–1 sodium chlonride with an ethanol yield of 4.66 ml dl^–1 (v/v) , SK-23 tolerated 6 g dl^–1 sodium chloride with an ethanol yield of 4.14 ml dl^–1 (v/v) and SK-26 showed resistance towards 8 g dl^–1 sodium chloride and give an ethanol yield of 3 ml dl^–1 (v/v).     

Direct isocratic normal-phase HPLC assay of fat-soluble vitamins and β-carotene in oilseeds

Non-conventional seeds are of current interest because their constituents have unique chemical properties and they may augment the supply of edible oils. The study of seed oils for their minor constituents is useful in order to use both oil and the minor components effectively. Fat-soluble vitamins (FSV) and pro-vitamin A (#-carotene) are of particular importance in nutrition. Although fatty acid profiles of the selected seeds have been reported, no data about their FSV composition are yet available. Black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.), and niger (Guizotia abyssinica Cass.) seed oils were extracted with two different solvents [n-hexane (H) or chloroform/methanol (CM) (2:1 v/v)] in a Soxhlet-extractor. An isocratic normal-phase high-performance liquid chromatography (NP-HPLC) method for FSV and #-carotene analysis using a Zorbax-Sil silica column and an ultraviolet (UV) detector was developed. Isooctane/ethylacetate (96:4 v/v) was used as the eluant for the simultaneous elution of vitamin E, while isooctane/2-propanol (99:1 v/v) was used to elute other FSV and #-carotene. Saponification of seed oil samples was not required. The seed oil or its diluted solution in the selected mobile phase was therefore directly injected onto the HPLC column. All tocopherol derivatives were identified in seed oil samples. The main constituents in black cumin and niger seed oils were !- and %-tocopherols, whereas #-tocopherol (575-672 µg g^-1 oil) was the major tocopherol in coriander seed oil. #-Carotene was measured in high levels in coriander seed oil (739-892 µg g^-1 oil), followed by niger seed oil (574-702 µg g^-1 oil) then black cumin (569-593 µg g^-1 oil). Examined seed oils, especially niger seed oil, were characterized by high amounts of phylloquinone (>0.2% of oil content). Retinoic acid, vitamin A-alcohol, vitamin D (D₂+D₃), and menadione (vitamin K₃) have not been detected in all oilseed samples. The aim of the present investigation was to estimate FSV and #-carotene content of studied oilseeds. The results obtained show that because of their specific FSV constitution, these seed oils are promising new oils.     

Selection of starter cultures for the production of ugba,a fermented soup condiment

Ugba is a traditional fermented African oil bean condiment that serves as flavouring in soups or as low-cost protein snack in Nigeria. A total of 56 strains of spore-forming bacteria were isolated from 21 ugba samples purchased from retail markets in southwestern Nigeria. Bacillus subtilis (26 strains) was identified as the dominant microorganism of the fermented samples. The total viable count for the ugba samples obtained from the retail outlets was in the range of 1.4᎒⁷-2.8᎒⁹ cfu/g, while the spore-forming bacteria had a count of 1.8᎒¹⁰ cfu/g. Strains of B. subtilis were selected as potential starter cultures based on their enzymatic activities and ability to produce stickiness. The nine selected strains had a range of scores of proteolytic activity (5.2-7.4 mm), amylolytic activity (4.4-5.6 mm) and stickiness (15.1-17.3 cm). Relative viscosity was in the range of 2.3-5.3 U/ml, while protease activity was from 30.1-51.4 U/ml. However, B. subtilis MM-4:B12 that had the highest scores was chosen as starter culture for the laboratory preparation of ugba, and a viable count of 1.4᎒¹⁰, pH of 8.1 and 46.5% moisture content were obtained at the end of 72 h fermentation period. The fermented product had the peculiar characteristics of the traditionally prepared sample which is usually fermented for about 5 days. There were no significant differences in the protein (17.8-18.1%), fat (40.9-41.2) and titratable acidity (0.11-0.13%) of both starter culture fermented ugba and those obtained from the retail markets. However, organoleptic evaluation scores showed the starter-inoculated samples being rated higher by the consumers with regard to consistency, aroma and taste.     

Application of lactic acid bacteria starter cultures for decreasing the biogenic amine levels in sauerkraut

Sauerkrauts from shredded white cabbage of six varieties were prepared in six laboratory experiments by initial fermentation at 22 °C for 14 days, then stored at 5-6 °C and analysed after 6 months. Seven biogenic amines were extracted with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Eight common sauerkraut quality parameters were also determined. In three experiments, a commercial strain of Lactobacillus plantarum and a mixed preparation of Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus, were applied at doses of 5᎒⁴, 1᎒⁵ and 5᎒⁵ CFU/g of cabbage. In three further experiments, L. plantarum, Microsil and a commercial strain of Lactobacillus buchneri were applied at doses of 5᎒⁵ and 5᎒⁶ CFU/g. Spontaneously fermented sauerkrauts were prepared as the control variants in all experiments. L. plantarum at doses of at least 5᎒⁵ CFU/g significantly (P<0.05) suppressed formation of putrescine, tyramine and cadaverine, amines occurring at the highest levels. Spermidine contents varied between 10 and 30 mg/kg and were not affected by the starter cultures. Levels of tryptamine, spermine and histamine were very low, often below the detection limits. For practical application, a dose of at least 5᎒⁶ CFU/g of the tested L. plantarum strain seems to be likely. The tested strains of L. buchneri and E. faecium showed tyrosine decarboxylase activity in an in vitro test.     

α-Amylases in raw and fermented African oil bean seeds (Pentaclethra macrophylla benth)

The !-amylases of raw and fermented seeds of African oil bean (Pentaclethra macrophylla Benth) were isolated, partially purified (tenfold) and subjected to different thermal and pH conditions. The specific activities of the purified enzyme from raw and fermented seeds were 0.037 ml^-1 min^-1 and 0.88 ml^-1 min^-1 respectively. The !-amylase from fermented seeds was more thermostable with optimum activity at 70 °C, compared to the optimum temperature of 60 °C obtained for the raw seed enzyme. The assayed !-amylases were stable over a wide range of pH (3.0-7.0), with optimum activity found at pH 6.0 and pH 5.0 for raw and fermented seeds respectively. The results show that the !-amylase of Pentaclethra macrophylla complements the microbial amylases in the mainly bacterial fermentation of African oil bean seeds to 'ugba', a highly nutritious food condiment. While the !-amylase in the raw seeds was basically the plant enzyme, that isolated from the fermented seeds was a combination of plant and microbial !-amylases.     

Determination of some phenolic compounds in flax seed and nettle roots by HPLC with coulometric electrode array detection

A method for determination of secoisolariciresinol, homovanillyl alcohol, p-coumaric acid, and ferulic acid in flax seed and nettle roots was developed. At first the naturally occurring esters and glycosides were cleaved by alkaline methanolysis (sodium methoxide) followed by enzymatic hydrolysis (cellulase) to get the aglycones. Then the compounds were isocratically separated (60 mM sodium acetate/ethanol/acetonitrile/glacial acetic acid =81:9:9:1, v:v:v:v, flow rate: 0.8 ml/min.) on a Spherisorb S-ODS 1 column (250 mm x 4.6 mm, i.d, particle size: 5 µm) using 7-hydroxy-4-methylcoumarin as internal standard and detected at eight electrodes in a coulometric electrode array detector. The potentials of the 1st to the 5th electrode were set to +150 to +350 mV ( in increments of 50 mV), the potentials of the 6th to the 8th electrode were adapted from +550 to +750 mV (in increments of 100 mV) against modified palladium reference electrodes. In six flax seed samples secoisolariciresinol was found in the range from 5,000 to 10,000 µg/g and the content of p-coumaric and ferulic acid varied between 100 and 450 µg/g. In nettle roots the content of the lignan is about 10 times lower than in flax seed.     

Kinetics of 3-chloropropane-1,2-diol (3-MCPD) degradation in high temperature model systems

A study of the decay kinetics of 3-chloropropane-1,2-diol (3-MCPD) at high temperatures in aqueous model systems is reported. Data obtained over the pH range 5-6.5 and temperatures of 80-142 °C agreed well with a predicative model derived from earlier kinetic data. The results showed that 3-MCPD was unstable in aqueous solutions approaching infinite dilution at temperatures above 80 °C. Comparison of the experimental decay of 3-MCPD with that based on the kinetic parameters A (=10^{7.73347+0.83775pH}s-1) and E_a (=119.2 kJ mol^-1) was good to within 20% over the temperature and pH ranged studied. A putative mechanism for the degradation reaction is discussed together with the implications for the decay and formation of 3-MCPD in food systems.     

Oil composition of coriander (<Emphasis Type="Italic">Coriandrum sativum </Emphasis>L.) fruit-seeds

Coriander (Coriandrum sativum L.) seeds were extracted with chloroform/methanol (2:1, v/v) and the amount of total lipid was 28.4% of seed weight. The major fatty acid was petroselinic acid (65.7% of the total fatty acid methyl esters) followed by linoleic acid. Chromatography on a silica column with solvent of increasing polarity yielded 93.0% neutral lipids, 4.14% glycolipids, and 1.57% phospholipids. Thin-layer chromatography on silica gel was used to obtain major neutral lipid subclasses. Fatty acid profile of neutral lipid subclasses, triacylglycerols, and sterol content were determined using gas-liquid chromatography. Six triacylglycerol molecular species were detected but one component (C54:3) corresponding to tripetroselinin, and/or dipetroselinoyl oleoyl glycerol comprised more than 50% of the total triacylglycerols. Sterol content was estimated to be at a high level (5186 µg g^-1 oil). Stigmasterol, #-sitosterol, ƕ-avenasterol, and campesterol were found to be the sterol markers. Phospholipid subclasses were separated by high-performance liquid chromatography on a silica column by gradient elution from isooctane/2-propanol (6:8, v/v) to isooctane/2-propanol/water (6:8:0.6, v/v/v) with detection at 205 nm. The major individual phospholipid subclasses were phosphatidylcholine followed by phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.     

Phenols and metals in sugar-cane spirits. Quantitative analysis and effect on radical formation and radical scavenging

A fluorescence-based analytical method for quantification of phenolic compounds in sugar cane spirits (and other distilled alcoholic beverages) was developed. Sample preparation involved reverse-phase solid phase extraction and separation by gradient reversed-phase HPLC with fluorescence detection. Twenty-one Brazilian sugar cane spirits (aged and non-aged cachaça) were analyzed and phenol, guaiacol, o-cresol, p-/m-cresol, 3, 5-xylenol, 4-ethylphenol, 4-ethylguaiacol, 2-ethylphenol, eugenol, (+)-catechin, (-)-epicatechin, and scopoletin quantified. The detection limit was between 0.01 mg l^-1 (eugenol and scopoletin) and 0.1 mg l^-1 [(+)-catechin and (-)-epicatechin]. Kaempferol and quercetin were quantified in the same spirits, together with copper and iron, using HPLC (spectrophotometric detection) and atomic absorption spectroscopy, respectively. Large variations between various spirits were noted: total phenols were between 1.5 and 70 mg l^-1, flavonoids were from below detection to 3.5 mg l^-1, Cu was between 0.04 and 7.0 mg l^-1; and Fe between 0.01 and 0.78 mg l^-1. The tendency of radical formation in the spirits was determined by electron spin resonance spectroscopy using N-t-butyl-!-phenylnitrone spin trapping, and radical scavenging capacity was determined spectrophometrically using the stable 2,2-diphenyl-1-picrylhydrazyl radical as probe. Radical formation depends mainly on the Cu content, while the radical scavenging and antioxidative capacity mainly depends on the flavonoid content. (+)-Catechin and (-)-epicatechin are most important for the antioxidative capacity as confirmed in a model experiment, where oxidation was induced by iron catalysis.     

Stereoselectivity of the β-lyase-catalyzed cleavage of <Emphasis Type="Italic">S</Emphasis>-cysteine conjugates of pulegone

Stereoisomers of 8-S-cysteinyl-p-menthan-3-one synthesized from (R)- and (S)- pulegone by Michael addition of cysteine were characterized by means of GC, GC-MS, LC-MS, and ¹H-NMR. Conjugates were treated with three sources of cysteine-S-conjugate #-lyase: (i) a crude enzyme extract prepared from Eubacterium limosum, (ii) tryptophanase from E. coli and (iii) yeast cells. The result was liberation of 8-mercapto-p-menthan-3-one, a powerful flavoring substance exhibiting a cassis-type odor note. The enzyme-catalyzed cleavage of the thio-ether bond proceeded with low enantioselectivity. Discrimination of diastereoisomers was more pronounced with a clear preference of the cis-configured substrates.     

Continuous production of wine vinegar in bubble column reactors of up to 60-litre capacity

This paper describes the development of a continuous acetic acid fermentation process for the production of wine vinegar in bubble column reactors of up to 60 l capacity. To determine appropriate fermentation conditions a study of the influence of residual ethanol concentration, inlet flow rate and aeration was carried out using a 6-l laboratory reactor, white table wine as fermentation medium, a temperature of 30 °C and an air flow rate of 0.125 min^-1 (vvm). The concentration of acetic acid obtained in the continuous wine vinegar production ranged from 91 g/l at 28.6 ml/h to 28 g/l at 154.1 ml/h by increasing the inlet flow rate. As expected, the biomass decreased as well, from 208 mg/l to 106 mg/l. The maximum acetification rate was observed in the range 85-110 ml/h, corresponding to a value of about 1.1 g/l/h. A further increase in the flow rate produced a slight decrease in the acetification rate. Best yields, between 94.5 and 94.7%, were obtained in the flow rate range of 60-75 ml/h. The acetification rate was improved only by about 10% by increasing the aeration from 0.125 to 0.250 min^-1. The continuous wine vinegar production was scaled up from the laboratory fermentor to a 60-l pilot acetator. During the steady state (residential time >6), with an inlet flow rate of 950 ml/h, temperature of 30 °C and aeration of 0.250 min^-1, the following parameters were obtained: acetic acid concentration 72 g/l, overall productivity 1.41 g/l/h and yield 94.2%.     

Antifungal activity of lauric acid derivatives against Aspergillus niger

The antifungal effects of lauric acid and four lauric acid derivatives (monolauroylglycerol, D-laurate A, T-laurate A, 6-O-lauroysucrose) were tested on the spore germination and the growth rate of Aspergillus niger DMF 0801. The results showed that the tested substances varied in their antifungal activity and they also confirmed the relation of the structure of tested substances and their antifungal effects. Monolauroylglycerol at concentration level between 0.2 and 1.8 mmol l^-1 caused the highest inhibition of spore outgrowths whereas 6-O-lauroysucrose at concentration between 0.05 and 1.8 mmol l^-1 did not exhibit inhibition of spore germination. The inhibition of colony growth rate was detected mainly at presence of 6-O-lauroysucrose.     

Photobacterium phosphoreum isolated as a luminescent colony from spoiled fish, cultured in model system under controlled atmospheres

A luminescent colony isolated from spoiled hake, tentatively identified as Photobacterium phosphoreum, was cultured in a sterile fish juice at 1ǃ °C. Growth, trimethylamine, off-odour and biogenic amines production under four gas mixtures (60% CO₂/15% O₂/25% N₂, 60% CO₂/40% O₂, 40% CO₂/60% O₂, 40% CO₂/40% O₂/20% N₂) and under air were tested. Aerobically incubated bacteria showed the highest counts and trimethylamine production after 43 days of storage (>10⁸ cfu/ml and 28 mg TMA-N/100 ml, respectively). Mixtures containing CO₂ and O₂ seemed to exert an inhibitory effect both on growth and trimethylamine production by this bacteria, specially for mixtures containing 60% CO₂. Off-odour production was detected when the bacteria was incubated in air from day 15. Cadaverine was the major amine produced by P. phosphoreum followed by histamine. Putrescine and spermidine production generally increased during the aerobic incubation. However, the level of histamine and tyramine was higher under the gas mixture containing 60% CO₂/15% O₂/25% N₂ than in air, whereas the agmatine production seemed to be enhanced under any gas mixture assayed.     

Chemical composition and microbiological changes during spontaneous and starter culture fermentation of Enam Ne–Setaakye, a West African fermented fish-carbohydrate product

Enam Ne-Setaakye, a fermented fish-carbohydrate meal was made from minced fish flesh (75%), yam (20.5%), onions(1%), ginger(1%) and salt(2.5%). A total of 113 isolates belonging to the genus Lactobacillus, Pediococcus, Bacillus, Pseudomonas, Klebsiella, Staphylococcus, Debaromyces and Candida were obtained from the natural fermentation process of the product. Lactobacillus brevis and Pediococcus pentosaceus were the dominant species with 24% and 19% occurrence respectively, while Candida species least occurred (3%).Changes in the viable count of the microflora showed the dominance of lactic acid bacteria from the 48 h of fermentation period (5.8᎒ cfu/g) to the end (2.3᎒ cfu/g). Enterobacteriaceae which was detected at a level of 10 cfu/g at 0 h, were not detectable after 72 h. Moulds were not isolated, while the yeast population became detectable from 72 h reaching 10 cfu/g at the end of fermentation. On the other hand, a shorter fermentation period of 72 h with a LAB count of 10 cfu/g, non-detection of enterobacteriaceae, moulds and a yeast population of 10 cfu/g were obtained when the fermentation was carried out using L. brevis and P. pentosaceus as starter cultures. A pH level of 4.3, 3.1% titratable acidity and 2.1% (^w/_wp) salt content were obtained in the starter culture fermented Enam Ne-Setaakye. There were significant differences in the moisture and free fatty acid contents of both naturally and starter culture fermented samples, but the protein content (16%) was not significantly different. Sensory evaluation of the products showed consumer preference for the steamed samples.     

Effects of an edible coating, fungicide and cold storage on microbial spoilage of cherries

The effects of an edible coating, i.e., Semperfresh^TM, fungicide, i.e., Imazalil, and cold storage on microbial spoilage of cherries were studied. Two group of cherries (Prunus avium) were coated just after harvest with 1% and 2% Semperfresh^TM fruit coating. Two other group of cherries were treated with 75 ppm and 150 ppm concentrations of Imazalil and then coated with a 1% concentration of Semperfresh^TM fruit coating. Half of the cherries from each of the above groups was stored at ambient temperature (~30Dž °C) and at 40-50% relative humidity, the other half of the cherries was stored at cold storage (0 °C) and at 95-98% relative humidity. Each group was analyzed for the mold and yeast count. The Semperfresh^TM edible coating resulted in a slightly increased fungal spoilage of fruits. Imazalil fungicide treatment clearly decreased and retarded the microbial spoilage of fruits. As a result, low temperature storage was the most effective solution for both microbial spoilage and delay in the ripening.     

Differentiation of natural and synthetic phenylacetic acids by 2H NMR of the derived benzoic acids

The origin of 11 samples of phenylacetic acid 4, including those microbially produced from L-phenylalanine 1 and obtained by hydrolysis of penicillin G, has been studied by natural abundance deuterium NMR spectroscopy. The analysis performed on 4 failed since the (D/H) values relative to the aromatic ring nuclei and to the methylene moieties of the analysed phenylacetic acid samples resulted as similar. The permanganate oxidation of 4 into benzoic acid 5 allowed a differentiation in the ²H NMR spectrum of the signals of the ortho, meta and para nuclei of the aromatic ring, which instead for phenylacetic acid collapse in a single signal. Considering the deuterium content of the different positions in the aromatic ring, the differentiation between the natural rather than synthetic origin of the skeleton of the examined products was possible. The deuterium content of the aromatic ring for the synthetic materials is near to the statistical distribution, whereas for the products of natural derivation it follows a para>ortho>meta enrichment.     

Identification and quantitation of flavonols in rowanberry (Sorbus aucuparia L.) juice

Eight flavonols were detected in rowanberry juice, six quercetin and two kaempferol glycosides which were identified as quercetin 3-O-galactoside, quercetin 3-O-glucoside, two quercetin 3-O-dihexosides, quercetin pentose-hexoside and quercetin hexose-pentoside and two kaempferol dihexosides. The total content of identified flavonols in the rowanberry juice was 291 mg L^-1. Quercetin dihexosides contributed 78% to the flavonol content. However, chlorogenic and neochlorogenic acid were the most abundant phenolic compounds in the rowanberry juice (544 and 273 mg L^-1, respectively). From a nutritional point of view, rowanberry juice provides 70 mg of flavonols and 196 mg of chlorogenic acids per serving. HPLC-MSⁿ and HPLC-DAD in combination with solid phase extraction were used to obtain these results.     

Linalool disaccharides as flavour precursors from green coffee beans (<Emphasis Type="Italic">Coffea arabica</Emphasis>)

Two linalool disaccharides, isolated from green coffee beans (Coffea arabica), were identified as 3(S)-linalool-3-O-#-D-glucopyranosyl-#-D-apiofuranoside and 3(S)-linalool-3-O-#-D-glucopyranosyl-!-L-arabinopyranoside. The structures were established by one- and two-dimensional ¹H and ¹³C NMR spectra as well as by ESI MS/MS spectra.     

Changes to polyphenols in the process of production of must and wines from blackcurrants and cherries. Part I. Total polyphenols and phenolic acids

The effect of pulp treatment on the qualitative and quantitative changes to polyphenol compounds exhibiting antioxidant properties, in musts and wines from blackcurrants and cherries was investigated. The following variants of pulp treatment were used: hot maceration, hot maceration and pulp pectinolysis with Rohapect MA Plus and Pektopol PM preparations, and also pulp pectinolysis with Rohapect and Pektopol preparations. Blackcurrant musts contained from 4800 to 6600 mg l^-1 of total polyphenols and cherry musts from 3060 to 3920 mg l^-1. The fermentation process caused a decrease in polyphenols content of approximately 25%. In production of fruit wines, the pulp treatment method had a considerable effect on the total content of polyphenols. In blackcurrant wines the highest extraction of polyphenols was obtained after pectinolysis with a Rohapect preparation. In the case of cherry wines the highest content of these compounds was extracted during pectinolysis with a Pektopol preparation. In musts and wines the presence of the following compounds, derivatives of hydroxycinnamic acid, was determined: neochlorogenic, chlorogenic, caffeic, p-coumaric and ferulic acids. The content of neochlorogenic acid was the highest both in musts and wines from blackcurrants and cherries and amounted to 41.7-126.3 mg l^-1, and 24.7-35.3 mg l^-1 respectively for blackcurrants and 74.3-87.7 mg l^-1and 44.5-71.4 mg l^-1 respectively for cherries. The enzymatic preparation Pektopol PM contained an enzyme acting as depsidase. It broke down chlorogenic acid and other compounds to simple phenolic acids (caffeic acid, p-coumaric acid).