Inhibition of oxidation of unsaturated fatty acid methyl esters by essential oils

The essential oils from 16 various spice plants were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids methyl esters isolated from linseed oil. The content of methyl oleate, methyl linoleate, and methyl linolenoate after 1, 2, and 4 months of autooxidation were used as criteria to estimate the antioxidant efficiencies of essential oils. In 4 months, 92% of the methyl linolenoate and 79% of the methyl linoleate were oxidized in a control sample of a model system. It was found that the most effective antioxidants were essential oils from clove bud, cinnamon leaves, and oregano. They inhibited autooxidation of methyl linolenoate by 76–85%. The antioxidant properties of these essential oils were due to phenols— eugenol, carvacrol, and thymol. Essential oil from coriander did not contain phenols, but it inhibited methyl linolenoate oxidation by 38%. Essential oils from thyme, savory, mace, lemon, and tea tree inhibited methyl linolenoate oxidation by 17–24%. The other essential oils had no antioxidant properties.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.     

Performance of rabbits and oxidative stability of muscle tissues as affected by dietary supplementation with Oregano essential oil

The effect of dietary supplementation with oregano essential oil on the performance of rabbits, and the susceptibility of the produced raw and thermally treated muscle tissue to lipid oxidation during refrigerated storage, were investigated. A total of 96 weaned rabbits were separated into four equal groups with three subgroups each. One group was given the basal diet and served as control, two groups were administered diets supplemented with oregano essential oil at levels of 100 and 200?mg/kg diet, whereas the remaining group was given a diet supplemented with α-tocopheryl acetate at 200?mg/kg. During the 42-day experimental period, body weight and feed intake were recorded weekly and the feed conversion ratio was calculated. Feeding the experimental diets to rabbits, performance parameters were not affected. Therefore, dietary oregano essential oil exerted no growth-promoting effect on rabbits. With increased supplementation of oregano essential oil, malondialdehyde values decreased in both raw and thermally treated muscles during refrigerated storage. This finding suggests that dietary oregano essential oil exerted a significant antioxidant effect. Dietary supplementation of oregano essential oil at the level of 200?mg/kg was more effective in delaying lipid oxidation compared with the level of 100?mg/kg, but inferior to dietary supplementation of 200?mg α-tocopheryl acetate per kg. This study indirectly provides evidence that antioxidant compounds occurring in oregano essential oil were absorbed by the rabbit and increased the antioxidative capacity of tissues.     

Performance of rabbits and oxidative stability of muscle tissues as affected by dietary supplementation with oregano essential oil

The effect of dietary supplementation with oregano essential oil on the performance of rabbits, and the susceptibility of the produced raw and thermally treated muscle tissue to lipid oxidation during refrigerated storage, were investigated. A total of 96 weaned rabbits were separated into four equal groups with three subgroups each. One group was given the basal diet and served as control, two groups were administered diets supplemented with oregano essential oil at levels of 100 and 200 mg/kg diet, whereas the remaining group was given a diet supplemented with alpha-tocopheryl acetate at 200 mg/kg. During the 42-day experimental period, body weight and feed intake were recorded weekly and the feed conversion ratio was calculated. Feeding the experimental diets to rabbits, performance parameters were not affected. Therefore, dietary oregano essential oil exerted no growth-promoting effect on rabbits. With increased supplementation of oregano essential oil, malondialdehyde values decreased in both raw and thermally treated muscles during refrigerated storage. This finding suggests that dietary oregano essential oil exerted a significant antioxidant effect. Dietary supplementation of oregano essential oil at the level of 200 mg/kg was more effective in delaying lipid oxidation compared with the level of 100 mg/kg, but inferior to dietary supplementation of 200 mg alpha-tocopheryl acetate per kg. This study indirectly provides evidence that antioxidant compounds occurring in oregano essential oil were absorbed by the rabbit and increased the antioxidative capacity of tissues.     

Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.     

白鱀豚额隆油的研究——Ⅰ.额隆油的脂肪酸组成

世界珍稀水兽白豚(Lipotes vexillifer.)是我国名贵特产之一,属齿鲸亚目(Odontoceti),淡水豚总科(Platanistoidea),白鱀豚科(Lipotidae)。白鱀豚生活在长江中、下游。借回声定位系统以探知外界情况(荆显英等,1981)。通常认为,在声发射过程中,齿鲸类饱含油分的额隆组织起着声透镜的作用。额隆的这种特殊生理机能,与其所含脂质的脂肪酸组成密切相关。1980年陆佩洪等报道了白鱀豚额隆油的酸价、碘价、皂化价、不皂化物及甘油三酯(比色法)的含量。有关白鱀豚额隆油的脂肪酸组成,迄今尚未研究。目前我们才开始进行该项工作。     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

Effect of oregano essential oil on the engraftment and development of Lewis carcinoma in F1 DBA C57 black hybrid mice

The effect of a low uptake dose of oregano essential oil with drinking water for three months (Origanum vulgare L.) on the degree of Lewis carcinoma engraftment and some parameters of oxidative stress has been studied in vivo using F1 DBA C57 Black hybrid mice. Oregano essential oil has been established to possess an anticancer activity. The degree of tumor engraftment decreased by 1.8 times, its size decreased by 1.5 times, and the development of tumor was significantly suppressed in sick mice under the effect of oregano essential oil. It was found that the uptake of essential oil did not affect the intensity of lipid peroxidation in the brain of mice and resulted in a significantly (by 36%) decreased content of secondary lipid oxidation products in the liver as shown in a reaction with thiobarbituric acid as compared to control subjects. The activity of antioxidant enzymes was found to increase after three months of essential oil uptake (by 1.5–3 times) as compared to the control group. This effect of essential oil supports the presence of bioantioxidant properties in this essential oil.     

Autooxidation of a mixture of lemon essential oils,methyl linolenoate,and methyl oleinate

Stability of components of a mixture of methyl linolenoate and methyl oleinate with two lemon (Citrus limon L.) essential oils in hexane during their autooxidation in light was studied by gas chromatography. The essential oils differed by their quantitative ratio of components: the single-fold (1x) oil contained approximately 90% monoterpene hydrocarbons and 1.47% citral, whereas the proportions of hydrocarbons and citral in the tenfold (10x) oil were approximately 60 and 18.32%, respectively. The concentration and composition of essential oils influence the rates of fatty-acid oxidation and fatty-acid peroxide cleavage. The 1x lemon oil inhibited the oxidation of methyl linolenoate and methyl oleinate, whereas the 10x oil accelerated these processes. The distinctions in the resistance of the major components of lemon essential oil to oxidation, which are determined by their composition and antioxidant properties of unsaturated fatty acids, were revealed.     

Effectiveness of cell-adsorbed bacteriocin produced by Lactobacillus curvatus CWBI-B28 and selected essential oils to control Listeria monocytogenes in pork meat during cold storage

Aims:  To study the effectiveness of a combination of cell-adsorbed bacteriocin (CAB; a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments) of a Lactobacillus curvatus strain with oregano or savory essential oil to control Listeria monocytogenes in pork meat at 4°C.
Methods and Results:  The antimicrobial activity of the CAB and six different essential oils was tested by the well diffusion assay against L. monocytogenes M, Escherichia coli 10536 and Salmonella serotype Typhi CWBI-H1. The anti- Listeria activity of the CAB and oregano or savory essential oils was also investigated in pork meat. The results of the well diffusion assay showed that CAB was only inhibitory to L. monocytogenes while savory and oregano essential oils were the most active against the three indicator bacteria. In pork meat, Listeria counts have declined from c. 10² CFU g⁻¹ to below the detectable limit during the first week of storage in samples treated with CAB or oregano essential oil and in those treated with CAB combined with oregano or savory essential oil. However, the counts of L. monocytogenes have increased after the third week of storage in all samples with the exception of those treated with the combination of CAB and oregano essential oil. The combination of CAB with savory essential oil resulted in a 2-week delay of the growth rebound compared with samples treated with CAB alone.
Conclusions:  Addition of oregano or savory essential oil exhibited a synergistic effect with CAB to control L. monocytogenes in pork meat during storage at 4°C.
Significance and Impact of the Study:  The combination of CAB with oregano or savory essential oil may be effectively used in meat industry to enhance the safety and stability of meat products.     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

Effect of oregano essential oil on microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres

AIMS: This study aimed to determine the combined effect of packaging (air, modified atmosphere) with or without the addition of essential oil not only on the selection of microbial association of meat but also to determine any significant difference in microbial metabolites produced from the prevailing bacteria. METHODS AND RESULTS: Samples of minced meat were mixed with different concentration of oregano essential oil (0, 0.05, 0.5 and 1% v/w) and packed under aerobic or with modified atmosphere (Mixed Gas Modified Atmosphere--MGMA, 40% CO2/30% N2/30% O2; or CO2 Modified Atmosphere--COMA, 100% CO2) and stored at 5 degrees C. In all packaging conditions, only concentrations of 0.5% and 1% oregano oil were effective. Inhibition was evident in the order air < MGMA < COMA. Oregano essential oil delayed glucose and lactate consumption aerobically as well as under MGMA. pH changes were also evident. Furthermore, proteolysis was significantly inhibited in aerobically stored samples, and so was the production of acetate under MAP. Similar results were obtained for the other organic acids eluted from HPLC column. CONCLUSIONS: Oregano essential oil delayed microbial growth and suppressed the final counts of the spoilage micro-organisms. It also caused a pronounced alteration in the physico-chemical properties of the minced meat. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial analysis alone as spoilage index may misrepresent the effect of a hurdle such as essential oils on spoilage.     

Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters

The transient rise in plasma triacylglycerol fatty acids after single-dose ingestion of fish oil as triacylglycerols, free acids, or ethyl esters with linseed oil as an absorption standard was used to determine the relative absorption of fish oil fatty acids in eight men. As free acids, the fish oil fatty acids were well absorbed (greater than or equal to 95%). As triacylglycerols, eicosapentaenoic acid (1.00 g) and docosahexaenoic acid (0.67 g) were absorbed only 68% and 57% as well as the free acids. The ethyl esters were absorbed only 20% and 21% as well as the free acids. The incomplete absorption of eicosapentaenoic and docosahexaenoic acids from fish oil triacylglycerols correlates well with known in vitro pancreatic lipase activity.     

Lipolysis of menhaden oil triacylglycerols and the corresponding fatty acid alkyl esters by pancreatic lipase in vitro: a reexamination

In order to distinguish between possible fatty acid differences during lumenal lipolysis and cellular absorption, we have reinvestigated the in vitro hydrolysis of menhaden oil and its alkyl esters by pancreatic lipase. For this purpose we incubated menhaden oil or its fatty acid methyl and ethyl esters with porcine pancreatic lipase in the presence of bile salts and determined the composition of the released free fatty acids, monoacylglycerols, diacylglycerols, and residual triacylglycerols, or the free fatty acids and residual alkyl esters, respectively, by thin-layer and gas-liquid chromatography. There was significant discrimination against the delta 4- to delta 7-unsaturated fatty acids of both medium and long chain lengths during the hydrolysis of menhaden oil and its fatty acid ethyl esters. In general, the ethyl esters were hydrolyzed 10-50 times more slowly than the corresponding glyceryl esters, depending on the exact ratio of the two substrate types. None of the triacylglycerols or ethyl esters, however, was completely resistant to hydrolysis resulting in an eventual cleavage of all the alkyl esters and presumably all the primary ester bonds in the triacylglycerol molecules. Since the rate of release of the least resistant fatty acid exceeded that of the most resistant acid by only a factor of 6, it is concluded that in the presence of a large excess of lipase the liberated fatty acids would approach the composition of the dietary alkyl or glyceryl esters, as observed during lumenal lipolysis (Yang, L.-Y., A. Kuksis, and J. J. Myher. 1989. Biochem. Cell Biol. 67: 192-204).     

Sugar ester synthesis by a mycelium-bound Mucor circinelloides lipase in a micro-reactor equipped with water activity sensor

The mycelium-bound Mucor circinelloides lipase was used for the synthesis of esters of saccharides and fatty acids in 37 ml reactor equipped with magnetic stirrer and water activity sensor. Either di-n-pentyl ether or the mixture of di-n-pentyl and petroleum ethers were applied as reaction media. Water activity sensor provided on-line monitoring of this parameter and control of continuous processes of ester synthesis. It was found that two natural antioxidants, i.e. carotene and astaxanthin activated this lipase in organic solvents that could be beneficial for the synthesis of esters of compounds sensitive to oxidation, e.g. polyunsaturated fatty acids.     

Intestinal absorption of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

The relative cellular uptake and incorporation into prechylomicrons and chylomicrons was investigated for the menhaden and rapeseed oil fatty acids, when given by stomach tube as the original oils or the corresponding methyl and ethyl esters. The intermediates and final products of cellular acylation were determined by chromatographic methods at various times over a period of 1-24 h. There was little selectivity in the uptake among the oligo- and poly-unsaturated fatty acids of menhaden oil, when either oil or esters were fed. In contrast, the long-chain saturated and monounsaturated fatty acids of rapeseed oil were discriminated against during both cellular uptake and reacylation (60% overall reduction in utilization). Also, there was detectable discrimination against the long-chain polyunsaturated monoacylglycerols of menhaden oil and against the long-chain saturated and monounsatured monoacylglycerols of rapeseed oil during both cellular uptake and reacylation (30% overall reduction in utilization). Evidence was obtained for an indiscriminate cellular uptake of variable amounts (4-22%) of intact dietary methyl and ethyl esters of fatty acids, which, however, appeared in the chylomicrons only to a very limited extent (0.1-1.0% of total lipid). During peak absorption the cellular and lymphatic appearance of fatty acids from the digestion and absorption of the alkyl esters was nearly 50% lower than that from the corresponding triacylglycerols. The slower absorption of the fatty acids from the alkyl ester feeding is hypothetically attributed to a lower efficiency of the phosphatidic acid pathway, which is required in the absence of dietary 2-monoacylglycerols, but other mechanisms cannot be excluded.     

Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters

The present study was undertaken to investigate the effect of n-9, n-6, and n-3 dietary fatty acid ethyl esters on basal (uninduced) and Fe2+/ascorbate (induced) lipid peroxidation (LPO) in salivary gland (SG) of mice. Feeding n-3 ethyl ester polyunsaturated fatty acids (PUFA) increased the uninduced and induced LPO in SG homogenates. In contrast, feeding olive oil ethyl esters (n-9) significantly lowered the induced and uninduced LPO in SG tissue. Salivary gland susceptibility to LPO increased in the order of: olive oil < corn oil < safflower oil < n-3 ethyl esters. Olive oil esters in the diet increased primarily the 18:1 levels in SG tissue. Whereas feeding n-3 PUFA notably increased the superoxide dismutase (SOD) and catalase activities in SG homogenates, no significant changes were seen between n-9 and n-6 PUFA-fed mice. Lower levels of Vitamin E (Vit E) in the tissues of n-3 PUFA-fed mice indicate that the higher the dietary lipid unsaturation, the higher the requirement for Vit E in the diet. Our results indicate that, similar to other organs, salivary gland susceptibility to uninduced or induced oxidation depends on the source of dietary PUFA. In conclusion, feeding olive oil increases the resistance of SGs to induced and uninduced LPO.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Ecophysiological attributes of Salmonella typhimurium in liquid culture and within a gelatin gel with or without the addition of oregano essential oil

The growth of Salmonella typhimurium in liquid culture and in a gel matrix system at two pH values (5.0 and 7.0) with (0.03% w/v) or without oregano essential oil was studied. It was shown that the type of growth media (liquid or gel) influenced significantly both the type of end-product formation and growth of bacteria as well as the inhibitory efficacy of the essential oil. The oil inhibited S. typhimurium more strongly in the liquid medium than in the gelatin matrix. In particular, the addition of essential oil in gelatin matrix delayed the initiation of growth and caused a slight suppression of the maximum population level, while in liquid culture, growth was prevented completely in identical conditions. Structure also was found to affect the rate of consumption of glucose and the rate of production of end products. Formic and acetic acids were produced in both systems, while an unidentified peak was formed only in broth samples.