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Applications for biotechnology: present and future improvements in lactic acid bacteria.

The lactic acid bacteria are involved in the manufacture of fermented foods from raw agricultural materials such as milk, meat, vegetables, and cereals. These fermented foods are a significant part of the food processing industry and are often prepared using selected strains that have the ability to produce desired products or changes efficiently. The application of genetic engineering technology to improve existing strains or develop novel strains for these fermentations is an active research area world-wide. As knowledge about the genetics and physiology of lactic acid bacteria accumulates, it becomes possible to genetically construct strains with characteristics shaped for specific purposes. Examples of present and future applications of biotechnology to lactic acid bacteria to improve product quality are described. Studies of the basic biology of these bacteria are being actively conducted and must be continued, in order for the food fermentation industry to reap the benefits of biotechnology.

Biotechnology↗

Preparation of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams by use of ice microparticulates.

Biodegradable foams of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) for tissue engineering were fabricated by a porogen-leaching technique using ice microparticulates as the porogen material. PLLA or PLGA solution in chloroform was mixed with ice microparticulates. The mixtures were frozen by being placed in molds in liquid nitrogen and freeze-dried to form the foams. Scanning electron microscopic observation of the PLLA and PLGA foams showed that evenly distributed and interconnected pore structures were formed in these foams. The porosity and surface area of the foams increased with an increase in the weight fraction of the ice microparticulates, while the median pore size remained unchanged. The pore structures of the foams could be manipulated by controlling processing variables such as the size and weight fraction of the ice microparticulates and polymer concentration.

Biocompatible Materials↗

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus.

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.

Carbohydrate Metabolism↗

Properties of multiphase microspheres of poly(dl-lactic acid) or poly(dl-lactic-co-glycolic acid) produced by mechanical agitation, sonication, or potentiometric dispersion.

The effect of processing techniques on the molecular weight of the polymer in biodegradable microspheres of poly(dl-lactic acid) (PLA) and poly(dl-lactic-co-glycolic acid) (PLGA) was investigated. Multiphase microspheres were produced by conventional agitation, potentiometric dispersion, or sonication. Gel permeation chromatography was used to determine the molecular weight of the polymer before and after processing. Polymer in microspheres of PLA produced by sonication experienced a 21% decrease in molecular weight of the polymer after 90 s of sonication, while microspheres made by potentiometric dispersion or mechanical agitation exhibited insignificant changes in molecular weight. Microspheres produced by potentiometric dispersion were found to have a more narrow particle size distribution compared to the other methods. A decrease in the internal diameter of the infusion tube used to produce multiphase microspheres by the potentiometric dispersion method was found to decrease the mean particle size of the resultant microspheres. Two surfactants were investigated at varied levels for utilization in the continuous phase, and particle size analysis revealed that increased surfactant levels caused an increase in the mean particle size of microspheres containing BSA produced by potentiometric dispersion. This phenomenon was attributed to an increase in conductivity of the continuous phase as the surfactant level was increased.

Chemistry, Pharmaceutical↗

Poly-L-lactic acid.

Poly-L-lactic acid, a synthetic biodegradable polymer, is available as an injectable intradermal implant for use in the correction and restoration of the signs of facial lipoatrophy in patients with HIV infection. In a 96-week noncomparative study in HIV-infected patients with severe facial lipoatrophy, 41% of patients treated with intradermal poly-L-lactic acid injections had a total cutaneous thickness of >10 mm at the nasogenian fold at week 24. Poly-L-lactic acid was injected into multiple sites of each cheek at baseline and at weeks 2, 4, and 6. Significant (p < 0.001) increases from baseline in total cutaneous thickness were reported at all evaluation time points up to 96 weeks after the start of treatment. In a randomized, nonblind study, significant (p < 0.001) increases from baseline in dermal thickness were observed at week 24 in HIV-infected patients with facial lipoatrophy who received immediate or deferred (by 12 weeks) treatment with poly-L-lactic acid, administered by injection into multiple intradermal sites every 2 weeks (three injection sessions in total). Improvements in depression and anxiety scores, patient visual analog scale assessments, and photographic assessments were also reported in patients treated with poly-L-lactic acid in the immediate versus deferred treatment trial. Poly-L-lactic acid was generally well tolerated. No serious adverse events were reported.

Adipose Tissue↗

Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture.

The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered.

Cells, Cultured↗

Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture.

Biodegradable foams of hydrophobic polymers can be efficiently wet by two-step immersion in ethanol and water, which overcomes the hindered entry of water into air-filled pores. Ethanol readily enters into the porous polymer, after which it is diluted and replaced by water. This method was evaluated for porous disks of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) foams of copolymer ratios 85:15 and 50:50. For PLLA disks of 0.88 porosity and 1730 microns thickness, prewetting with ethanol for 1 h increased the percentage of void volume filled with water after 48 h from 23 to 79%. The same enhanced entry of water was also observed for prewet PLGA 85:15 disks of 0.86 porosity and 1300 microns thickness, which exhibited an increase from 59 to 97% void volume occupied by water. Furthermore, the water entry even after 1 h was very close to its plateau value for all prewet polymers tested. In recent studies, this method has been useful in uniformly seeding three-dimensional biodegradable polymer substrates for cell and tissue culture.

Culture Techniques↗

Effect of dietary sucrose in humans on blood uric acid, phosphorus, fructose, and lactic acid responses to a sucrose load.

10 men and 9 women consumed diets that were identical, except for the 30% of the calories derived from either starch or sucrose, for 6 weeks in a crossover design. Of the total calories, 10% were given at breakfast and 90% at dinner. A sucrose load of 2 g/kg body weight was administered 1 week before and during the last week of both dietary periods. Blood was drawn before (fasting) and 0.5, 1, 2 and 3 h after the sucrose load. Serum uric acid was significantly higher (p < 0.01) and blood fructose significantly lower (p < 0.05) before and at all times after the sucrose load when subjects consumed the sucrose versus the starch diet. Levels of serum inorganic phosphorus and blood lactic acid were not affected by kind of carbohydrate in the diet.

Adult↗

Lactic acid bacteria inducing a weak interleukin-12 and tumor necrosis factor alpha response in human dendritic cells inhibit strongly stimulating lactic acid bacteria but act synergistically with gram-negative bacteria.

The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G-) commensals, such as members of the family Enterobacteriaceae, may, however, be immunomodulators that are as important as G+ organisms but tend to be overlooked. Dendritic cells (DCs) are crucial immune regulators, and therefore, the present study aimed at investigating differences among human gut flora-derived LAB and G- bacteria in their patterns of DC polarization. Human monocyte-derived DCs were exposed to UV-killed bacteria, and cytokine secretion and surface marker expression were analyzed. Profound differences in the DC polarization patterns were found among the strains. While strains of LAB varied greatly in their capacity to induce interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha), G- strains were consistently weak IL-12 and TNF-alpha inducers. All strains induced significant amounts of IL-10, but G- bacteria were far more potent IL-10 inducers than LAB. Interestingly, we found that when weakly IL-12- and TNF-alpha-inducing LAB and strong IL-12- and TNF-alpha-inducing LAB were mixed, the weakly IL-12- and TNF-alpha-inducing LAB efficiently inhibited otherwise strong IL-12- and TNF-alpha-inducing LAB, yet when weakly IL-12- and TNF-alpha-inducing LAB were mixed with G- bacteria, they synergistically induced IL-12 and TNF-alpha. Furthermore, strong IL-12- and TNF-alpha-inducing LAB efficiently up-regulated surface markers (CD40, CD83, CD86, and HLA-DR), which were inhibited by weakly IL-12- and TNF-alpha-inducing LAB. All G- bacteria potently up-regulated surface markers; however, these markers were not inhibited by weakly IL-12- and TNF-alpha-inducing LAB. These much divergent DC stimulation patterns among intestinal bacteria, which encompass both antagonistic and synergistic relationships, support the growing evidence that the composition of the gut flora affects immune regulation and that compositional imbalances may be involved in disease etiology.

Bifidobacterium↗

Lactic acid fermentation of cassava dough into agbelima.

The souring of cassava dough during fermentation into the fermented cassava meal, agbelima, was investigated. Four different types of traditional inocula were used to ferment the dough and increases in titrable acidity expressed as lactic acid from 0.31-0.38 to 0.78-0.91% (w/w) confirmed the fermentation to be a process of acidification. The microflora of all inocula and fermenting dough contained high counts of lactic acid bacteria, 10(8)-10(9) cfu/g in all inocula and 10(7)-10(8), 10(8)-10(9) and 10(9) cfu/g at 0, 24 and 48 h in all fermentations. Lactobacillus plantarum was the dominant species of lactic acid bacteria during all types of fermentation accounting for 51% of 171 representative isolates taken from various stages of fermentation. Other major lactic acid bacteria found were Lactobacillus brevis, 16%, Leuconostoc mesenteroides, 15% and some cocci including Streptococcus spp. whose numbers decreased with fermentation time. The lactic acid bacteria were responsible for the souring of agbelima through the production of lactic acid. All L. plantarum, L. brevis and L. mesenteroides isolates examined demonstrated linamarase as well as other enzymatic activities but did not possess tissue degrading enzymes like cellulase, pectin esterase and polygalacturonase. The aroma profile of agbelima did not vary with the type of inoculum used and in all samples the build-up of aroma compounds were dominated by a non-identified low molecular weight alcohol, 1-propanol, isoamyl alcohol, ethyl acetate, 3-methyl-1-butanol and acetoin. Substantial reductions occurred in the levels of cyanogenic compounds present in cassava during fermentation into agbelima and detoxification was enhanced by the use of inoculum.

Colony Count, Microbial↗

Influence of medium composition on lactic acid production from dried whey by Lactobacillus delbrueckii.

The production of lactic acid, lactic acid dehydrogenase (LDH) and single cell protein (SCP) from dried whey by fermentation with Lactobacillus delbrueckii subsp, bulgaricus 369 was investigated. A medium consisting of dried whey and supplements which enabled exponential growth of the organism was developed. Maximum lactic acid production was achieved using enriched whey (8% w/v), at pH 5.5 for 24 h. Addition of yeast extract plus MnCl2 gave the highest production of lactic acid (1.71 g/l.h), specific activity of LDH (1.03 U/mg protein) and SCP (32.8%). In batch culture, the addition of 20 g/l glucose plus 0.75 g N/l, as (NH4)2SO4, achieved the highest enzymatic activity. By contrast, when xylose and cellobiose were used as a source of fermentable carbohydrates all variables were repressed.

Animals↗

Comparing HPLC and UV spectrophotometric analysis methods for determining the stability of sorbic acid in nonionic creams containing lactic acid.

This paper describes a comparison between ultraviolet (UV) spectrophotometric and high-performance liquid chromatographic (HPLC) methods of analysis for the determination of sorbic acid in nonionic creams containing lactic acid. Sorbic acid is an antimycotic agent and is used as a preservative in pharmaceuticals, cosmetics, and food products. UV spectrophotometric analysis was done by calculating the concentration of remaining sorbic acid from the absorbance values and the molar extinction coefficient EM258 = 24,080. A decrease in absorbance at 258 nm was accompanied by a simultaneous increase in total carbonyls and monoaldehyde content and the appearance of a very weak absorption maximum between 215 and 225 nm. HPLC analysis was done with a Hypersil BDS C8 column with detection at 254 nm and employing a mobile phase consisting of a mixture of buffer and methanol (7:3 v/v) at a pH of 2.25. The buffer consisted of 0.85% H2SO4 in 17.5 mM KH2PO4. The validation results, together with statistical treatment of the data, demonstrated the reliability of both procedures. A drawback of the UV methods was, however, its lack of adequate measurement of sorbic acid stability at higher temperatures. For these assays, the HPLC method was found to be adequate, and it should therefore be used to obtain accurate stability data for sorbic acid in creams.

Chromatography, High Pressure Liquid↗