Probiotic Yeasts and How to Find Them-Polish Wines of Spontaneous Fermentation as Source for Potentially Probiotic Yeasts.

One approach towards maintaining healthy microbiota in the human gastrointestinal tract is through the consumption of probiotics. Until now, the majority of probiotic research has focused on probiotic bacteria, but over the last few years more and more studies have demonstrated the probiotic properties of yeast, and also of species besides the well-studied Saccharomyces cerevisiae var. boulardii. Probiotic strains have to present the ability to survive in harsh conditions of the host body, like the digestive tract. Must fermentation might be an example of a similar harsh environment. In the presented study, we examined the probiotic potential of 44 yeast strains isolated from Polish wines. The tested isolates belonged to six species: Hanseniaspora uvarum, Pichia kluyveri, Metschnikowia pulcherrima, Metschnikowia ziziphicola, Saccharomyces cerevisiae and Starmerella bacillaris. The tested strains were subjected to an assessment of probiotic properties, their safety and their other properties, such as enzymatic activity or antioxidant properties, in order to assess their potential usefulness as probiotic yeast candidates. Within the most promising strains were representatives of three species: H. uvarum, M. pulcherrima and S. cerevisiae. H. uvarum strains 15 and 16, as well as S. cerevisiae strain 37, showed, among other features, survivability in gastrointestinal tract conditions exceeding 100%, high hydrophobicity and autoaggregation, had no hemolytic activity and did not produce biogenic amines. The obtained results show that Polish wines might be a source of potential probiotic yeast candidates with perspectives for further research.

Inherently acidic films based on chitosan lactate-doped starches and pullulan as carries of nisin: A comparative study of controlled-release and antimicrobial properties.

Nisin (NIS) Z was incorporated (0.05 %, 0.1 %, 0.2 %) into edible films based on chitosan lactate (CHL) and 75/25 blends of polysaccharides (corn starch (CS), wheat starch (WS), oxidized potato starch (OPS), pullulan (PUL)) with CHL. The increase in the NIS/polymer ratio promoted the diffusion-driven release. Compared with the fully dissolvable CHL and PUL/CHL carriers, the starch/CHL films had limited solubility (≈27-37 %) and, consequently, ensured slower/incomplete release of NIS. The assayable NIS half-release times, determined in water, ranged from <1 min to ∼13 h. Probably due to the similar pH (≈4.5), there were generally no large differences between the antibacterial activities of the formulations. The NIS-supplemented systems limited the growth of some pathogens (B. cereus, L. monocytogenes, S. aureus), phytopathogens (P. carotovorum), and bacterial starter cultures. The NIS improved the UV-blocking ability of the films, but the 0.2 % NIS addition weakened (by ≈17-32 %) the tensile strength of most of the films.

Reduction of the Fusarium Mycotoxins: Deoxynivalenol, Nivalenol and Zearalenone by Selected Non-Conventional Yeast Strains in Wheat Grains and Bread.

Mycotoxins, toxic secondary metabolites produced by fungi, are important contaminants in food and agricultural industries around the world. These toxins have a multidirectional toxic effect on living organisms, causing damage to the kidneys and liver, and disrupting the functions of the digestive tract and the immune system. In recent years, much attention has been paid to the biological control of pathogens and the mycotoxins they produce. In this study, selected yeasts were used to reduce the occurrence of deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA) produced by Fusarium culmorum, F. graminearum, and F. poae on wheat grain and bread. In a laboratory experiment, an effective reduction in the content of DON, NIV, and ZEA was observed in bread prepared by baking with the addition of an inoculum of the test yeast, ranging from 16.4% to 33.4%, 18.5% to 36.2% and 14.3% to 35.4%, respectively. These results indicate that the selected yeast isolates can be used in practice as efficient mycotoxin decontamination agents in the food industry.

Biological Control of Fusarium culmorum, Fusarium graminearum and Fusarium poae by Antagonistic Yeasts.

The genus Fusarium is considered to be one of the most pathogenic, phytotoxic and toxin-producing group of microorganisms in the world. Plants infected by these fungi are characterized by a reduced consumer and commercial value, mainly due to the contamination of crops with mycotoxins. Therefore, effective methods of reducing fungi of the genus Fusarium must be implemented already in the field before harvesting, especially with alternative methods to pesticides such as biocontrol. In this study we identified yeasts that inhibit the growth of the pathogenic fungi Fusarium culmorum, F. graminearum and F. poae. Tested yeasts came from different culture collections, or were obtained from organic and conventional cereals. The greater number of yeast isolates from organic cereals showed antagonistic activity against fungi of the genus Fusarium compared to isolates from the conventional cultivation system. Cryptococcus carnescens (E22) isolated from organic wheat was the only isolate that limited the mycelial growth of all three tested fungi and was the best antagonist against F. poae. Selected yeasts showed various mechanisms of action against fungi, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores or production of extracellular lytic enzymes: chitinase and ß-1,3-glucanase. Of all the investigated mechanisms of yeast antagonism against Fusarium, competition for nutrients and the ability to inhibit spore germination prevailed.

Probiotic and Potentially Probiotic Yeasts-Characteristics and Food Application.

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Besides the well-known and tested lactic acid bacteria, yeasts may also be probiotics. The subject of probiotic and potentially probiotic yeasts has been developing and arising potential for new probiotic products with novel properties, which are not offered by bacteria-based probiotics available on the current market. The paper reviews the first probiotic yeast Saccharomyces cerevisiae var. boulardii, its characteristics, pro-healthy activities and application in functional food production. This species offers such abilities as improving digestion of certain food ingredients, antimicrobial activities and even therapeutic properties. Besides Saccharomyces cerevisiae var. boulardii, on this background, novel yeasts with potentially probiotic features are presented. They have been intensively investigated for the last decade and some species have been observed to possess probiotic characteristics and abilities. There are yeasts from the genera Debaryomyces, Hanseniaspora, Pichia, Meyerozyma, Torulaspora, etc. isolated from food and environmental habitats. These potentially probiotic yeasts can be used for production of various fermented foods, enhancing its nutritional and sensory properties. Because of the intensively developing research on probiotic yeasts in the coming years, we can expect many discoveries and possibly even evolution in the segment of probiotics available on the market.

Spicy Herb Extracts as a Potential Improver of the Antioxidant Properties and Inhibitor of Enzymatic Browning and Endogenous Microbiota Growth in Stored Mung Bean Sprouts.

The quality and shelf life of sprouts can be improved by postharvest application of water herb extracts. The effect of water infusions of marjoram, oregano, basil, and thyme on the phenolic content, antioxidant potential, and the microbiological and consumer quality of stored mung bean sprouts was studied. Compared to the control, the treatments increased total phenolic content. The highest amounts were determined in sprouts soaked in the thyme extract (6.8 mg/g d.m.). The infusions also inhibited the activity of enzymes utilizing phenolics, and marjoram and oregano were found to be the most effective. The increase in the level of phenolics was reflected in enhanced antioxidant properties (ability to quench cation radical ABTS•+, reducing and chelating power). Both total phenolics and flavonoids, as well as antioxidant capacities, were highly bioaccessible in vitro. All the natural extracts effectively reduced the growth of total mesophilic bacteria, coliforms, and molds (they were more effective than ascorbic and kojic acids). The treatments did not exert a negative influence on the sensory properties or nutritional value of the sprouts, and even improved starch and protein digestibility. These results are very promising and may suggest a wider used of natural extracts as preservatives of minimally processed food.

Effect of Basil Leaves and Wheat Bran Water Extracts on Antioxidant Capacity, Sensory Properties and Microbiological Quality of Shredded Iceberg Lettuce during Storage.

The effect of basil leaf (BLE) and wheat bran (WBE) extracts (potent anti-browning agents), on the phenolic content, antioxidant potential, microbiological quality, and consumer quality of shredded lettuce during storage were studied. Treatment of lettuce with increasing concentrations of BLE proportionally increased the total phenolic content and antioxidant properties. Compared to the control, the treatment enhanced the antiradical properties. This was especially visible during the analysis of the chemical extracts, while this effect was not retained in the potentially bioaccessible fraction. In the lettuce stored for 8 days, the highest reducing potential and ability to quench radicals were observed in samples treated with 1% BLE-33 mg Trolox equivalent/g d.m. and 2.8 mg Trolox equivalent/g d.m., respectively. Compounds exhibiting antiradical properties were easily bioaccessible in vitro. There was no negative effect of the treatments on the consumer quality. Most importantly, after 8 days of storage, lettuce treated with the studied extract, except 10% WBE, had higher microbiological quality. After 8-day storage, the coliforms count was reduced by 84% and 88% in samples treated with 0.5% BLE and 10% WBE, respectively. In conclusion, treatments of shredded lettuce with BLE and WBE maintain or even improve its quality during storage.

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level.

The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level-genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

Genomic and Proteomic Characterization of Bacteriophage BH1 Spontaneously Released from Probiotic Lactobacillus rhamnosus Pen.

Lactobacillus rhamnosus Pen is a human endogenous strain used for the production of probiotic formula, which is effective in the prevention of antibiotic-associated diarrhoea. Our study showed that this probiotic strain releases bacteriophage BH1 without the addition of any inducing agent. Our research revealed that phage BH1 has a circular genome with a length of 40721 nt and a GC content of 44.8%. The genome of phage BH1 possesses 57 open reading frames which could be divided into functional modules associated with DNA packaging, morphogenesis, lysis, integration, genetic switch, and replication. In spite of similarity in morphology and genomic organization, comparative analysis revealed substantial genetic diversity and mosaic genomic architecture among phages described for the Lactobacillus casei group. Additionally, qPCR and ddPCR analysis confirmed earlier microscopic observations indicating that L. rhamnosus Pen liberates bacteriophage particles during growth. This occurs spontaneously, and is not a result of external inducing factors. For samples collected after 4 and 24 h of L. rhamnosus Pen culture, the number of attB and attP copies increased 2.5 and 12 times, respectively. This phenomenon, by introducing resistance to other phages or enhancing the biofilm-forming capabilities, may increase the survivability of microorganisms in their natural ecological niche. Conversely, spontaneous phage induction may be an important virulence factor for bacteria, posing a potential threat for the human host.

Lactobacillus plantarum 299V improves the microbiological quality of legume sprouts and effectively survives in these carriers during cold storage and in vitro digestion.

Probiotics improve consumers' health and additionally may positively influence the microbiological and organoleptic quality of food. In the study, legume sprouts were inoculated with Lactobacilllus plantarum 299V to produce a new functional product ensuring the growth and survival of the probiotic and high microbiological quality of the final product. Legume sprouts, which are an excellent source of nutrients, were proposed as alternative carriers for the probiotic. The key factors influencing the production of probiotic-rich sprouts include the temperature (25°C) of sprouting and methods of inoculation (soaking seeds in a suspension of probiotics). Compared to the control sprouts, the sprouts enriched with the probiotic were characterized by lower mesophilic bacterial counts. In the case of fresh and stored probiotic-rich sprouts, lactic acid bacteria (LAB) accounted for a majority of total microorganisms. The Lb. plantarum population was also stable during the cold storage. The high count of LAB observed in the digest confirmed the fact that the studied sprouts are effective carriers for probiotics and ensure their survival in the harmful conditions of the digestive tract in an in vitro model. Enrichment of legume sprouts with probiotics is a successful attempt and yields products for a new branch of functional foods.

Improvement of Candida parapsilosis by genome shuffling for the efficient production of arabitol from l-arabinose.

Arabitol is used in the food industry as a low-calorie sweetener. It is produced by yeasts during the biotransformation process of l-arabinose. Genome shuffling was performed in Candida parapsilosis DSM 70125, an efficient producer of arabitol, to obtain fusants with improved arabitol production ability. Four mutants from the parental library were used for the first round of genome shuffling. The best fusants, GSI-1 and GSI-10A, were subjected to a second round of genome shuffling. Finally, two fusants, GSII-3 and GSII-16, produced concentrations of arabitol that were 50% higher than that of the wild-type strain during selection culture. Under the optimal conditions established for C. parapsilosis, the two fusants produced 11.83 and 11.75 g/L of arabitol and were approximately 15-16% more efficient than the wild-type strain. Flow cytometry analysis showed that the ploidy of the new strains did not change.

The Ability of a Novel Strain Scheffersomyces (Syn. Candida) shehatae Isolated from Rotten Wood to Produce Arabitol.

Arabitol is a polyalcohol which has about 70% of the sweetness of sucrose and an energy density of 0.2 kcal/g. Similarly to xylitol, it can be used in the food and pharmaceutical industries as a natural sweetener, a texturing agent, a dental caries reducer, and a humectant. Biotechnological production of arabitol from sugars represents an interesting alternative to chemical production. The yeast Scheffersomyces shehatae strain 20BM-3 isolated from rotten wood was screened for its ability to produce arabitol from L-arabinose, glucose, and xylose. This isolate, cultured at 28°C and 150 rpm, secreted 4.03 ± 0.00 to 7.97 ± 0.67 g/l of arabitol from 17-30 g/l of L-arabinose assimilated from a medium containing 20-80 g/l of this pentose with yields of 0.24 ± 0.00 to 0.36 ± 0.02 g/g. An optimization study demonstrated that pH 4.0, 32°C, and a shaking frequency of 150 rpm were the optimum conditions for arabitol production by the investigated strain. Under these conditions, strain 20BM-3 produced 6.2 ± 0.17 g/l of arabitol from 17.5 g/l of arabinose after 4 days with a yield of 0.35 ± 0.01 g/g. This strain also produced arabitol from glucose, giving much lower yields, but did not produce it from xylose. The new strain can be successfully used for arabitol production from abundantly available sugars found in plant biomass.

The occurrence of killer activity in yeasts isolated from natural habitats.

Yeast's ability to restrict the growth and kill other yeasts, fungi and bacteria has been known for over 50 years. Killer activity was detected in yeasts deposited in the world collections or isolated from natural habitats. In this study, isolates from the forest environment, leaves of fruit trees, flower petals, cereals and frozen fruit have been screened in terms of their killer activities. Killer activity was tested on strains belonging to six yeast species: Candida, Rhodotorula, Pichia, Pachysolen, Yarrowia, Trichosporon. The reference strains were Kluyveromyces lactis Y-6682 and Kluyveromyces marxinanus Y-8281, well-known to be sensitive to yeast killer toxins. Among one hundred and two tested strains, 24 (23.5% of isolates) showed positive killer action, and 10 (9.8% of the isolates) a weak killer action against at least one sensitive reference strain. The highest killer activity was observed among isolates from forest soil and flowers.

Characterization of films based on chitosan lactate and its blends with oxidized starch and gelatin.

Minimal inhibitory concentration (MIC) of chitosan lactate (CHL) was tested against bacteria and phytopathogenic fungi. Then, the structural, physicochemical and antimicrobial properties of films based on CHL, oxidized potato starch (OPS), and gelatin (GEL) were investigated. With the exception of Rhizopus nigricans, CHL was effective against the target organisms. Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) were more sensitive to CHL than Gram-negative bacteria (Pectobacterium carotovorum and Escherichia coli). Cryo-SEM images showed total miscibility between the polymers in the blends and the ATR-FTIR spectra revealed that there was an interaction among the polymeric components. Pure CHL films displayed the highest moisture content (25.51%), water vapor permeability (48.78gmmm(-2)d(-1)kPa(-1)), and the lowest tensile and puncture strength (2.00 and 1.45MPa, respectively) among the studied films. CHL50/GEL50 films had lower permeability, higher mechanical strength, and lower elongation compared to CHL50/OPS50 films. Films obtained from CHL and CHL50/GEL50 were completely water-soluble and did not show sorbitol recrystallization. The incorporation of CHL into OPS and GEL films did not affect their transparency and improved UV-blocking capacity. CHL films were the only ones that exhibited antibacterial efficiency. Antifungal activities against Alternaria alternata and Monilinia fructigena were detected for CHL and CHL50/GEL50 films.

Optimization of arabitol production by karyoductant SP-K 7 of S. cerevisiae V30 and P. stipitis CCY 39501 using response surface methodology.

L-arabitol is used in the food and pharmaceutical industries. It can be secreted by genetically modified Saccharomyces cerevisiae carrying the genes responsible for pentose metabolism in yeast cells. The process of the biotransformation of L-arabinose to arabitol is highly dependent on culture conditions. The aim of this investigation was to use statistical response surface methodology (RSM) for optimization of biotransformation of L-arabinose to arabitol by a karyoductant of S. cerevisiae V30 and Pichia stipitis CCY 39501, named SP-K7. Batch cultures of yeast were performed according to a Plackett-Burman design, and three factors, rotation speed, L-arabinose concentration, and temperature, were chosen for a central composite design (CCD) applied in order to optimize the production of the polyol by the karyoductant. On the basis of results obtained using 20 combinations of batch cultures of karyoductant SP-K7, the optimal levels of the factors were determined as: rotation speed 150 rpm, concentration of L-arabinose 32.5 g/l, and temperature 28 degrees C. In such conditions, the predicted concentration of arabitol after two days of incubation of SP-K7 should be 18.367 g/l. The value of R2 = 0.93195 suggested that this model was well-fitted to the experimental data. A verification of the model in experimental conditions confirmed its usefulness.

Application of response surface methodology to enhancement of biomass production by Lactobacillus rhamnosus E/N

Response surface methodology (RSM) was employed to study the effects of various medium components on biomass production by Lactobacillus rhamnosus E/N. This strain is commonly used in the pharmaceutical and food industries due to its beneficial effect on the human gut and general health. The best medium composition derived from RSM regression was (in g/l) glucose 15.44, sodium pyruvate 3.92, meat extract 8.0, potassium phosphate 1.88, sodium acetate 4.7, and ammonium citrate 1.88. With this medium composition biomass production was 23 g/l of dry cell weight after 18 h of cultivation in bioreactor conditions, whereas on MRS the yield of biomass was 21 g/l of dry cell weight. The cost of 1 g of biomass obtained on MRS broth was calculated at the level of 0.44 € whereas on the new optimal medium it was 25 percent lower. It may be concluded then, that the new medium, being cheaper than the control MRS allows large scale commercial cultivation of the L. rhamnosus strain. This study is of relevance to food industry because the possibility to obtain high yield of bacterial biomass is necessary step in manufacturing of probiotic food.

The influence of selected prebiotics on the growth of lactic acid bacteria for bio-yoghurt production.

BACKGROUND: Prebiotics are a category of nutritional com-pounds grouped together, not necessarily by structural similarities, but by ability to promote the growth of spe-cific beneficial (probiotic) gut bacteria. Fructooligosaccharides (FOS) and inulin are among the most famous prebiotic compounds. In order to improve viability of probiotic bacteria during storage, fermented food should be supplemented with prebiotics. MATERIAL AND METHODS: Yoghurts were produced from skimmed milk powder and prebiotics (FOS, inulin or resistant starch), which were added at concentrations of 1%, 2% and 3%. Yoghurts were stored in +4°C for three weeks. Every week each kind of fermented drink was examined in order to check the growth of lactic acid bacteria. Apparent viscosity and texture of bio-yoghurt were determinated during refrigerated storage. RESULTS: The FOS and inulin addition to yoghurt caused an increase in the numbers of all bacteria in comparison to control yoghurt obtained without addition of prebiotics. The viable counts of Str. thermophilus, Lb. acidophilus and Bifidobacterium sp. when 1% of FOS was added to yoghurt were about 9 log cfu/g, 7.8 log cfu/g and 7.7 log cfu/g, respectively. In the presence of 1% of inulin, streptococci and bifidobacteria reached the growth at the level 8.8 log cfu/g and 7.5 respectively. Hardness and adhesiveness of yoghurt obtained with addition resistant starch increased systematically during 21 days of refrigerated storage. CONCLUSIONS: The numbers of lactic acid bacteria in obtained bio-yoghurts were sufficient in 97% of samples (10(6)-10(9) cfu/g) according to FAO/WHO protocols. Generally, viability of bacteria was sufficient for 14 days and then their numbers decreased but usually not below 10(6) cfu/g. Prebiotics as FOS and inulin added to bio-yoghurt exhibited stimulatory effect on growth Lb. acidophilus and Bifidobacterium sp. Addition of prebiotics caused an increase in apparent viscosity and hardness (in case of FOS) and decrease in syneresis of obtained bio-yoghurts.

Application of response surface methodology to enhancement of biomass production by Lactobacillus rhamnosus E/N.

Response surface methodology (RSM) was employed to study the effects of various medium components on biomass production by Lactobacillus rhamnosus E/N. This strain is commonly used in the pharmaceutical and food industries due to its beneficial effect on the human gut and general health. The best medium composition derived from RSM regression was (in g/l) glucose 15.44, sodium pyruvate 3.92, meat extract 8.0, potassium phosphate 1.88, sodium acetate 4.7, and ammonium citrate 1.88. With this medium composition biomass production was 23 g/l of dry cell weight after 18 h of cultivation in bioreactor conditions, whereas on MRS the yield of biomass was 21 g/l of dry cell weight. The cost of 1 g of biomass obtained on MRS broth was calculated at the level of 0.44 € whereas on the new optimal medium it was 25% lower. It may be concluded then, that the new medium, being cheaper than the control MRS allows large scale commercial cultivation of the L. rhamnosus strain. This study is of relevance to food industry because the possibility to obtain high yield of bacterial biomass is necessary step in manufacturing of probiotic food.

The Plackett-Burman design in optimization of media components for biomass production of Lactobacillus rhamnosus OXY.

The central composite design was developed to search for an optimal medium for the growth of Lactobacillus rhamnosus OXY. The effect of various media components, such as carbon sources, simple and complex nitrogen sources, mineral agents, and growth factors (vitamins B, amino acids) was examined. The first-order model based on Plackett-Burman design showed that glucose, sodium pyruvate, meat extract and mineral salts significantly influenced the growth of the examined bacteria. The second-order polynomial regression confirmed that maximum biomass production could be achieved by the combination of glucose (12.38 g/l), sodium pyruvate (3.15 g/l), meat extract (4.08 g/l), potassium phosphate (1.46 g/l), sodium acetate (3.65 g/l) and ammonium citrate (1.46 g/l). The validation of the predicted model carried out in bioreactor conditions confirmed the usefulness of the new medium for the culture of L. rhamnosus OXY in large scale. The optimal medium makes the culture of the probiotic bacterium L. rhamnosus OXY more cost effective.

Optimization of medium composition for enhancing growth of Lactobacillus rhamnosus PEN using response surface methodology.

Response surface methodology was used to optimize media components such as carbon and nitrogen (simple and complex) sources, mineral agents and growth factors (B vitamins, amino acids) for enhancing the biomass production of Lactobacillus rhamnosus PEN. For screening experiment the following carbon sources were selected: glucose, glucose+pyruvate, glucose+citrate, glucose+lactate, galactose, fructose, lactose, sucrose, maltose, lactulose, fructooligosaccharides, maltodextrins DP 4-7 and DP 13-17. Nitrogen sources such as yeast extract, meat extract and peptone K were used in lower concentrations than in MRS medium which served as a control. All experiments were run at 37 degrees C for 24-48 h under stationary conditions. Constituents chosen after the first screening experiments were further screened by the Plackett-Burman design. Glucose and sodium pyruvate, meat extract, potassium phosphate, sodium acetate, and ammonium citrate were chosen as promising medium components for further optimization studies. By solving the regression equation and analyzing the response surface carton, optimal concentrations of the components were determined as: glucose (13.4 g/l), sodium pyruvate (3.4 g/l), meat extract (7.2 g/l), potassium phosphate (2.0 g/I), sodium acetate (5.0 g/1) and ammonium citrate (2.0 g/l). In comparison to MRS broth the optimal medium contained fewer ingredients and in modified amounts but Lb. ihamnosus PEN showed better growth activity. Biomass concentration (as dry cell weight) of bacteria cultivated in optimal medium at bioreactor conditions was 5.5 g/l after 16 h of incubation, being higher in comparison with bacterial growth in MRS medium (1.9 g/l) under the same conditions. Moreover, the new medium was less expensive.