Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization

The cryotolerance of Lactobacillus delbrueckii ssp. bulgaricus is weak during vacuum freeze-drying. Many factors affect cryoresistance of these bacteria, such as cryoprotectant composition, the lyophilization technology used, and the intrinsic characteristics of the bacteria. In this research, we explored the fermentation technology and other preconditioning treatments of cells in improving the cryoresistance of Lactobacillus delbrueckii ssp. bulgaricus strains during lyophilization. The addition of yeast extract in the propagation medium exerted a negative effect on the cryotolerance of these bacteria and decreased survival during lyophilization. The count of the freeze-dried cells from medium containing a high level (4%) of yeast extract was only 4.1 × 10⁹ cfu/g, indicating a death rate as high as 88%, compared with the culture medium without yeast extract, with a lower death rate of 44.7%. When Lactobacillus delbrueckii ssp. bulgaricus ND02 was propagated in yeast extract-free de Man, Rogosa, and Sharpe broth at a set pH value of 5.1, the cells showed unexpectedly higher survival after freeze-drying. Viable counts of the lyophilized cell of strain ND02 cultivated at pH 5.1 could reach 1.05 × 10¹¹ cfu/g and survival of the freeze-drying process was 68.3%, whereas at pH 5.7, survival was only 51.2%. We also examined the effects of pretreatment of cells on survival of the bacteria after vacuum freeze-drying. By analyzing the effect of pretreatment conditions on the expression of cold- and heat-shock genes, we established 2 pretreatments that improved survival of cells after lyophilization. Optimal fermentation conditions and pretreatment of the cell-cryoprotectant mixture at 10°C for 2 h or 37°C for 30 min improved the cryoresistance of 4 strains of Lactobacillus delbrueckii ssp. bulgaricus to varying degrees. Cells of IMAU20269 and IMAU20291 that were pretreated showed enhanced survival of 16.06 and 16.82%, respectively, after lyophilization. Expression of cold- and heat-shock genes for pretreated strains ND02, IMAU80423, IMAU20269, and IMAU20291 was analyzed by using quantitative PCR. From the expression of 2 cold shock-induced genes (cspA and cspB) and 6 heat shock-induced genes (groES, hsp, hsp20, hsp40, hsp60, and hsp70), strain ND02 showed a higher relative quantity of gene expression and displayed superior resistance to cold-induced stress during the freeze-drying process.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Production of conjugated linoleic acid by Propionibacterium freudenreichii

Two strains each of Propionibacterium freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii were tested for their ability to produce conjugated linoleic acid (CLA) in sodium lactate medium (SLM), De Man-Rogosa-Sharpe (MRS) medium and skim-milk. Data showed that both strains were able to produce CLA in three media supplemented with different concentrations of sunflower oil. Maximum production of CLA (78.8 μg/ml) was observed after 36 h of incubation in MRS containing 12 mg/ml of sunflower oil by P. freudenreichii ssp. shermanii. Moreover, the growths of both strains were inhibited by sunflower oil and a positive relationship between CLA production and ability to tolerate sunflower oil was observed. At the same time, it was also observed that the inhibitory effects on P. freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii in three media follow the order SLM > skim-milk > MRS and SLM > MRS > skim-milk, respectively. Micro aerobic conditions were in favour of increasing the amounts of CLA. The amounts of CLA increased from 0 to 36 h under micro aerobic conditions and no significant (p > 0.05) increases in total CLA levels were observed after 80 h of incubation. Results showed that P. freudenreichii may have potential for producing CLA.     

Fatty acid profile, trans-octadecenoic, α-linolenic and conjugated linoleic acid contents differing in certified organic and conventional probiotic fermented milks

Development of dairy organic probiotic fermented products is of great interest as they associate ecological practices and benefits of probiotic bacteria. As organic management practices of cow milk production allow modification of the fatty acid composition of milk (as compared to conventional milk), we studied the influence of the type of milk on some characteristics of fermented milks, such as acidification kinetics, bacterial counts and fatty acid content. Conventional and organic probiotic fermented milks were produced using Bifidobacterium animalis subsp. lactis HN019 in co-culture with Streptococcus thermophilus TA040 and Lactobacillus delbrueckii subsp. bulgaricus LB340. The use of organic milk led to a higher acidification rate and cultivability of Lactobacillus bulgaricus. Fatty acids profile of organic fermented milks showed higher amounts of trans-octadecenoic acid (C18:1, 1.6 times) and polyunsaturated fatty acids, including cis-9 trans-11, C18:2 conjugated linoleic (CLA-1.4 times), and α-linolenic acids (ALA-1.6 times), as compared to conventional fermented milks. These higher levels were the result of both initial percentage in the milk and increase during acidification, with no further modification during storage. Finally, use of bifidobacteria slightly increased CLA relative content in the conventional fermented milks, after 7 days of storage at 4 °C, whereas no difference was seen in organic fermented milks.     

Complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starch-fatty acid mixtures

Effects of the type and amount of fatty acid (0-2.0 mmol/g-starch of lauric, myristic, palmitic, stearic, oleic, and linoleic acids) on the complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starch-fatty acid mixtures were investigated. Complex index (CI) evaluated by the reduction in the iodine binding capacity of starch increased with an increase in the amount of fatty acids, and reached a plateau depending on the type of fatty acid. The maximum CI value was higher in the order of lauric (49.9%), linoleic (47.6%), myristic (42.4%), oleic (36.7%), stearic (35.3%), and palmitic acid (30.9%). From the calorimetric study, it was demonstrated that melting temperature of the complexes was higher in the order of stearic (96.7 °C) > lauric, myristic, palmitic, and oleic (89.6-92.1 °C) > linoleic acid (78.3 °C). Melting enthalpy for complexes was roughly related to the CI value (R² = 0.667). From the in-vitro digestibility measurement, it was found that a certain amount of fatty acid reduced the starch content hydrolyzed at a given condition. Among them, 0.50 mmol/g-starch lauric and oleic acid samples showed the largest reduction in the hydrolyzed starch content. This result was related to the extent of complex formation characterized by CI value and its helical order characterized by melting temperature. In addition, there was a possibility of the complex formation between amylose and unsaturated fatty acid during the hydrolysis of gelatinized starch.     

A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii subspecies bulgaricus CFL1

The mechanisms of cellular damage that lactic acid bacteria incur during freeze-thaw processes have not been elucidated to date. Fourier transform infrared spectroscopy was used to investigate in situ the lipid phase transition behavior of the membrane of Lactobacillus delbrueckii ssp. bulgaricus CFL1 cells during the freeze-thaw process. Our objective was to relate the lipid membrane behavior to membrane integrity losses during freezing and to cell-freezing resistance. Cells were produced by using 2 different culture media: de Man, Rogosa, and Sharpe (MRS) broth (complex medium) or mild whey-based medium (minimal medium commonly used in the dairy industry), to obtain different membrane lipid compositions corresponding to different recovery rates of cell viability and functionality after freezing. The lipid membrane behavior studied by Fourier transform infrared spectroscopy was found to be different according to the cell lipid composition and cryotolerance. Freeze-resistant cells, exhibiting a higher content of unsaturated and cyclic fatty acids, presented a lower lipid phase transition temperature (Ts) during freezing (Ts = −8°C), occurring within the same temperature range as the ice nucleation, than freeze-sensitive cells (Ts = +22°C). A subzero value of lipid phase transition allowed the maintenance of the cell membrane in a relatively fluid state during freezing, thus facilitating water flux from the cell and the concomitant volume reduction following ice formation in the extracellular medium. In addition, the lipid phase transition of freeze-resistant cells occurred within a short temperature range, which could be ascribed to a reduced number of fatty acids, representing more than 80% of the total. This short lipid phase transition could be associated with a limited phenomenon of lateral phase separation and membrane permeabilization. This work highlights that membrane phase transitions occurring during freeze-thawing play a fundamental role in the cryotolerance of Lb. delbrueckii ssp. bulgaricus CFL1 cells.     

Effects of dietary replacement of fish oil by conjugated linoleic acid on some meat quality traits of Pacific white shrimp Litopenaeus vannamei

This experiment was conducted to evaluate the effects of fish oil replacement by conjugated linoleic acid (CLA) on body proximate analysis and thiobarbituric acid-reactive substances, fat content, shear force and fatty acid composition in musculature of Pacific white shrimp (Litopenaeus vannamei). Graded levels of CLA (0%, 0.5%, 1% and 2%) were added to the basic diet of shrimp at the expense of fish oil. Results showed that fat content (p = 0.036) and shear force (p = 0.001) in shrimp musculature were enhanced with increasing dietary CLA inclusion. Fish oil replacement by CLA significantly promoted the incorporation of cis-9, trans-11 CLA (p = 0.0001) and trans-10, cis-12 CLA (p < 0.0001) into shrimp musculature; moreover, the polyunsaturated fatty acid was elevated (p = 0.020) and monounsaturated fatty acid was reduced by CLA inclusion (p = 0.024). It was concluded that replacement of fish oil by CLA could improve some meat quality traits of shrimp and 1% CLA was an appropriate amount.     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

Evaluation and characterisation of Citrullus colocynthis (L.) Schrad seed oil: Comparison with Helianthus annuus (sunflower) seed oil

The physicochemical properties, fatty acid, tocopherol, thermal properties, ¹H NMR, FTIR and profiles of non-conventional oil extracted from Citrullus colocynthis (L.) Schrad seeds were evaluated and compared with conventional sunflower seed oil. In addition, the antioxidant properties of C. colocynthis seed oil were also evaluated. The oil content of the C. colocynthis seeds was 23.16%. The main fatty acids in the oil were linoleic acid (66.73%) followed by oleic acid (14.78%), palmitic acid (9.74%), and stearic acid (7.37%). The tocopherol content was 121.85 mg/100 g with γ-tocopherol as the major one (95.49%). The thermogravimetric analysis showed that the oil was thermally stable up to 286.57 °C, and then began to decompose in four stages namely at 377.4 °C, 408.4 °C, 434.9 °C and 559.2 °C. The present study showed that this non-conventional C. colocynthis seed oil can be used for food and non-food applications to supplement or replace some of the conventional oils.     

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Fat and fatty acids of white lupin (Lupinus albus L.) in comparison to sesame (Sesamum indicum L.)

The study was undertaken to compare fat and fatty acid profiles in white lupin (Lupinus albus ssp. albus) and sesame (Sesamum indicum L.), representing two different families, Fabaceae and Pedaliaceae. Fat levels were 10.74% and 55.44% in seeds of white lupin and sesame, respectively. The results indicated that oleic, linolenic and arachidic acids in seed fat were higher in white lupin than in sesame cultivars. Oleic acid was the predominant fatty acid in white lupin, whereas linoleic acid was predominant in sesame. Fat content (%) was statistically significantly correlated with linoleic, linolenic and arachidic acids at the genotypic level. The fatty acid composition of white lupin is useful for human consumption. Although oil content of white lupin was lower than that of sesame, white sweet lupin could be improved.     

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 °C for the free and 60 °C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kJ/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver–Burk plots and the V_max and K_m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%.     

Antioxidant activity of Spirulina platensis extracts by supercritical carbon dioxide extraction

Supercritical CO₂ extraction of antioxidants from Spirulina platensis was optimized using response surface methodology. About 10.26 g/kg of extracts from S. platensis could be obtained under the optimum conditions of 48 °C at 20 MPa over a 4 h period. The antioxidant activity of the extracts prepared under the optimized condition, determined by linoleic acid peroxidation inhibition method, was lower compared with BHT and Trolox, but significantly higher than α-tocopherol in 300 min and became similar to α-tocopherol after that. The components of the extracts were further analyzed, and the results showed that the extracts contained 85.1 g/kg of flavonoids, 77.8 g/kg of β-carotene, 113.2 g/kg of vitamin A and 3.4 g/kg of α-tocopherol, which may contribute greatly to their high antioxidant activity. The main fatty acids in the extracts were palmitic acid (35.32%), linolenic acid (21.66%) and linoleic acid (20.58%).     

Fatty acids of neutral and phospholipids of three endangered trout: Salmo trutta caspius Kessler, Salmo trutta labrax Pallas and Salmo trutta macrostigma Dumeril

Total (TL), neutral (NL) and phospholipid (PL) amounts and fatty acid (FA) composition of female Salmo trutta caspius, Salmo trutta labrax and Salmo trutta macrostigma were investigated during one year. Twenty-three FAs were identified in both NLs and PLs. The principal FAs of both fractions were palmitic acid in saturated fatty acid, oleic acid in monounsaturated fatty acid, docosahexaenoic acid (DHA) in n-3 polyunsaturated fatty acids (n-3 PUFAs) and linoleic acid in n-6 PUFAs. The highest values for TLs, NLs and PLs were found in winter. As a general trend, the highest n-3/n-6 ratios and eicosapentaenoic acid (EPA) + DHA amounts were found in the winter and this coincided with the lowest gonado-somatic index.     

Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. longifolia

This study was designed to evaluate antimicrobial and antioxidant activities of the essential oil and methanol extract from Mentha longifolia ssp. longifolia. The essential oil showed strong antimicrobial activity against all 30 microorganisms tested whereas the methanol extract almost remained inactive. In contrast, the extract showed much better activity than the essential oil in antioxidant activity assays employed, e.g. in the inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene/linoleic acid systems. In the former, the extract was able to reduce the stable free radical DPPH with an IC₅₀ of 57.4 μg/ml while that of the oils was 10 700 μg/ml. When compared to BHT, a synthetic antioxidant, both showed weaker antioxidative potential. Similarly, in β-carotene/linoleic acid assay, these samples were not effectively able to inhibit the linoleic acid oxidation; exhibiting only 24% and 36% inhibitions at 2 mg/ml, respectively; both were far below than that of BHT. Total phenolic constituent of the extract was 4.5 g/100 g as gallic acid equivalent. GC–MS analysis of the oil resulted in the identification of 45 constituents, cis-piperitone epoxide, pulegone and piperitenone oxide being the main components.     

Some low homogenization pressures improve certain probiotic characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K

Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus salivarius ssp. thermophilus, and Lactobacillus acidophilus are dairy cultures widely used in the manufacture of cultured dairy products. Commonly used homogenization pressures in the dairy industry are 13.80 MPa or less. It is not known whether low homogenization pressures can stimulate bacteria to improve their probiotic characteristics. Objectives were to determine the effect of homogenization at 0, 3.45, 6.90, 10.34, and 13.80 MPa on acid tolerance, bile tolerance, protease activity, and growth of L. delbrueckii ssp. bulgaricus LB-12, S. salivarius ssp. thermophilus ST-M5, and L. acidophilus LA-K. The cultures were individually inoculated in cool autoclaved skim milk (4°C) and homogenized for 5 continuous passes. Growth and bile tolerance of samples were determined hourly for 10 h of incubation. Acid tolerance was determined every 20 min for 120 min of incubation. Protease activity was determined at 0, 12, and 24 h of incubation. All homogenization pressures studied improved acid tolerance of L. delbrueckii ssp. bulgaricus LB-12 but had no beneficial effect on protease activity and had negative effects on growth and bile tolerance. A pressure of 6.90 MPa improved acid tolerance, bile tolerance, and protease activity of S. salivarius ssp. thermophilus ST-M5, but none of the homogenization pressures studied had an effect on its growth. Homogenization pressures of 13.80 and 6.90 MPa improved acid tolerance and bile tolerance, respectively, of L. acidophilus LA-K but had no effect on protease activity and its growth. Some low homogenization pressures positively influenced some characteristics of yogurt culture bacteria and L. acidophilus LA-K. Culture pretreatment with some low homogenization pressures can be recommended for improvement of certain probiotic characteristics.     

Direct quantification of fatty acids in dairy powders with special emphasis on trans fatty acid content

In this study a validated procedure for accurate determination of fatty acids in dairy products, with special emphasis on total trans fatty acids (TFA) content is presented. Dairy fat naturally contains 4–6% of trans fatty acids, mainly trans-octadecenoic acids (i.e. vaccenic acid), and 0.3–1.5% of conjugated linoleic acids (CLA). The proposed procedure does not require lipid extraction, and transesterification of lipids could be carried out directly on dairy products. Optimal analytical conditions have been developed to allow accurate determination of TFA content without prior fractionation of cis/trans FAME isomers by thin-layer chromatography. The methodology requires the use of a highly polar open tubular capillary column having at least 100 m length. CLA and other fatty acids from C4:0 (butyric) acid to long-chain polyunsaturated fatty acids (LC-PUFAs) could also be analyzed. Therefore, the methodology presented is versatile and could be used for both targeted analysis (e.g. determination of TFA in dairy products) and determination of the broad fatty acid profile in dairy products.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.