Studies On The Screening, Physiological And Metabolic Properties Of Leuconostoc Stains Used In Dairy Industry

Leuconostoc spp. are gram-positive and heterofermentative lactic acid bacteria found in raw foodstuffs, dairy products and vegetable fermentations. The microorganisms are able to convert citric acid from media into diacetyl, one important compound contributing to the aroma of dairy products like butter, cheeses or buttermilk. Moreover, Leuconostoc strains have found commercial applications as one of the most important starter cultures in dairy and vegetable processing, food preservation and even medical industry. The objectives of the present study were: (1) to isolate and screen leuconostoc strains from various sources like raw milks and fermented sauerkrauts; (2) to investigate their physiological and citrate-metabolizing properties under controlled conditions; (3) to obtain new leuconostoc strains available for the commercial preparation of starters used in dairy products. The following are main results.The three selective media HP, LUSM and LMM were used to isolate 45 strains of Leuconostoc spp. from raw cow milk, goat milk and fermented vegetables obtained form Inner Mongolia and Baoding City of Hebei Province based on vancomycin-resistant, citrate-fermenting and dextran-producing from saccharose. Among 45 isolates, 20 isolates were identified as Leuconostoc species based on the morphological, gram-staining, physiology and carbohydrate fermentations with reference to the type strain Leuconostoc mesenteriodies subsp. mesenteriodies 6055. Out of 20 leuconostoc species, 10 strains were identified as Leuconostoc mesenteriodies subsp. mesenteriodies, 4 strains as Leuconostoc mesenteriodies subsp. dextranicum, 1 strain as Leuconostoc mesenteriodies subsp. cremoris, 1 strain as Leuconostoc lactic, and 4 strains as Leuconostoc prarmesenteriodies.L. mesenteriodies subsp. mesenteriodies DM 1-2, L .mesenteriodies subsp. dextranicum WZ4-3 and L.prarmesenteriodies WZ-9-2, along with L.mesenteriodies subsp. mesenteriodies 6055, were tested for further studies according to their citrate-metabolizing and the amount of formed diacetyl. Compared to strain 6055 used as the control, the effects of glucose, saccharose and lactose on the cell growth and acidifying ability of strains DM 1-2, WZ4-3 and WZ-9-2 were carried out. The results showed that glucose and saccharose stimulated the growth and acidifying activity of strains 6055, DM 1-2 and WZ-9-2 very well, whereas saccharose was the optimal sugar supporting the cell growth of strain WZ4-3.Peptone, tryptone and casein-hydrolysate were utilized well as nitrogen sources by the strains6055. DM1-2, WZ4-3 and WZ-9-2. The cell growth and acidifying activity of 4 strains studied showed that no significant differences were found among the utilization of 3 nitrogen resources used as growth-promoting factors.Inclusion of citrate into MRS media incubated at 37°C, to some extent, reduced the acidifying activity of Leuconostoc strains 6055, DM 1-2, WZ4-3 and WZ-9-2, Moreover, addition of 0.15% citric acid to MRS media had not give a clear stimulation to the cell growth of 6055, DM1-2 and WZ-9-2 except strain WZ4-3.The effects of metal irons like Mn2+, Mg2* or Mn2+ plus Mg2+ on the cell growth and acidifying ability of Leuconostoc strains DM 1-2, WZ4-3 and WZ-9-2 were carried out. The results indicated that Mn2+ improved clearly the growth and acidifying activity of 4 strains tested, whereas Mg~" produced no clear stimulation to the growth and activity of leuconostoc strainsFour strains of Leuconostoc tested grew at a wide temperature range from 21°C to 32°C. Cell growth and acidifying activity showed that strains 6055 and DM 1-2 grew very well at 32°C. Strains WZ4-3 and WZ-9-2 had the largest growth and acidifying activity at 25°C. It was clear that the optimal cell growth and activity were strain-dependent.The activities of diacetyl reductase and lactic dehydrogenase (LDH) of the strains 6055, DM 1-2, WZ4-3 and WZ-9-2 were investigated based on the changes of the two enzymes in pH from 4.6 to 6.6. It was seen that the diacetyl reductase of strain 6055 showed a change in the shape of "v" (i.e. firstly declining, then reaching the lowest, and finally rising) as pH increased. When pH was 5.5, the level of diacetyl reductase from strain 6055 achieved the minimum having 0.290U. Its LDH activity decreased from 0.188U to 0.083 U when pH was raised. The levels of diacetyl reductase and LDH from strain DM 1-2 decreased gradually with increase in pH. When pH changed between 6.6 and 4.6, the enzyme's activity of strain DM 1-2 declined from 0.291U to 0.195U in diacetyl reductase or from 0.173U to 0.062U in LDH. Data from the present study showed that the activities of diacetyl reductase and LDH of strains WZ4-3 varied in the shape of "a" as pH rose from 4.6 to 6.6. The activity of its diacetyl reductase had a maximal value of 0.231U at pH 4.8; and its LDH reached maximum with 0.165 at pH 5.8. The diacetyl reductase of.strain WZ-9-2 increased slowly between 0.059U and 0.149U as pH declined from 6.6 to 4.6. Its LDH activity showed a decline from 0.278U to 0.114U when pH varied between 6.6 and 4.6.The colorimetric estimation of ONPG was used to determine [i-galactosidase activity of the four ieuconostoc strains 6055, DM 1-2, WZ4-3 and WZ-9-2. The levels of (3-galactosidase was 6.8x 10~'U for strain 6055, 8.4x 10"3U for strain DM1-2,2.9x I0~3U for strain WZ4-3 and 9.3x 10~2U for strain WZ-9-2, respectively. It was clear that the levels of |3-galactosidase formed by leuconostoc depended upon strains used.Steam-distillation-spectrophotometric method was used to deal with citrate utilization and subsequent diacetyl formation by the Leuconostoc strains DM 1-2 and WZ4-3 under different fermentation conditions. The results showed that addition of 0.15% citric acid to milk before inoculation increased the level of diacetyi from I.571ug/mL to 2.186|ag/mL produced by strain...