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.     

Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

Linoleic acid Isomerase Activity in Enzyme Extracts from Lactobacillus Acidophilus and Propionibacterium Freudenreichii ssp. Shermanii

Enzyme extracts from Lactobacillus acidophilus (CCRC14079) and Propionibacterium freudenreichii ssp. shermanii (CCRC11076) were reacted with linoleic acid at 50 °C for 10 min at pH 5, 6, 7, and 8, and the levels of conjugated linoleic acid (CLA) formation were determined by high performance liquid chromatography. CLA formations were observed in all the reactions catalyzed by the retentates using ultrafiltration membranes with 100 kDa nominal molecular weight cutoff, indicating the presence of the activity of linoleic acid isomerase with nominal molecular weight higher than 100 kDa in the retentates from two cultures. More CLA was formed at pH 5 of L. acidophilus treatment and t10c12, c11t13, and c9t11 CLA were 3 major CLA isomers produced. Results demonstrate a potential for CLA production through linoleic acid isomerase.     

Probiotic in lamb rennet paste enhances rennet lipolytic activity, and conjugated linoleic acid and linoleic acid content in Pecorino cheese

Cheeses manufactured using traditional lamb rennet paste, lamb rennet paste containing Lactobacillus acidophilus, and lamb rennet paste containing a mix of Bifidobacterium lactis and Bifidobacterium longum were characterized for the lipolytic pattern during ripening. Lipase activity of lamb rennet paste, lamb rennet containing Lb. acidophilus, and lamb rennet containing a mix of bifidobacteria was measured in sheep milk cream substrate. Rennet paste containing probiotics showed a lipase activity 2-fold greater than that displayed by traditional rennet. Total free fatty acid (FFA) in sheep milk cream was lower in lamb rennet paste (981 μg/g of milk cream) than in lamb rennet containing Lb. acidophilus (1,382.4 μg/g of milk cream) and in lamb rennet containing a mix of bifidobacteria (1,227.5 μg/g of milk cream) according to lipase activity of lamb rennet paste. The major increase of FFA in all cheeses occurred during the first 30 d of ripening with the greatest values being observed for C16:0, C18:0 C18:1. At 60 d of ripening all cheeses showed a reduction in the amount of free fatty acids; in particular, total free fatty acids underwent a decrease of more than 30% from 30 to 60 d in cheeses manufactured using traditional lamb rennet paste, whereas the same parameter decreased 10% in cheeses manufactured using lamb rennet paste containing Lb. acidophilus and cheeses manufactured using lamb rennet paste containing a mix of B. lactis and B. longum. Cheese containing Lb. acidophilus was characterized by the greatest levels of total conjugated linoleic acids (CLA) 9-cis, 11-trans CLA and 9-trans, 11-trans CLA, whereas cheese containing bifidobacteria displayed the greatest levels of free linoleic acid. Rennet pastes containing viable cells of Lb. acidophilus and a mix of B. lactis and B. longum were able to influence the amount of FFA and CLA in Pecorino cheese during ripening.     

Effect of incubation conditions and possible intestinal nutrients on cis-9, trans-11 conjugated linoleic acid production by Lactobacillus acidophilus F0221

The purpose of this study was to optimise the incubation conditions for cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) production by Lactobacillus acidophilus F0221 and evaluate the effect of possible intestinal nutrients on c9, t11 CLA production. Growth in Mann Rogosa Sharp broth supplemented with cysteine and containing 0.5 g L⁻¹ linoleic acid at pH 6.5 with anaerobic incubation at 37 °C for 40 h were found to be the optimal conditions for CLA production. Galactosaccharide, arabinogalactan, galactose and glucose had a higher promoting effect on CLA production (96.19–123.89 μg mL⁻¹) than other carbohydrates. Acetic acid had a higher promoting effect on CLA production (130.98 μg mL⁻¹) than other short chain fatty acids. These results provide detailed parameters for the production of c9, t11 CLA by L. acidophilus F0221 and are valuable for further understanding of c9, t11 CLA formation in the human intestine by this strain.     

Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin

A gas chromatographic procedure was used for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic; trans-10, cis-12 octadecadienoic; and trans-9, trans-11-octadecadienoic (c9t11, t10c12, t9t11) produced by lactobacilli. Four different cultures, two strains each of Lactobacillus acidophilus and Lactobacillus casei were tested for their ability to produce CLA from free linoleic acid in MRS broth supplemented with linoleic acid. Different concentrations of linoleic acid (0, 0.05, 0.1, 0.2 and 0.5 mg/ml) were added to MRS broth, inoculated with the lactobacilli, and incubated at 37 degrees C. Viable counts and amounts of individual isomers of CLA (c9t11, t10c12, t9t11) were measured at 0, 24, 48, and 72 h. All the cultures were able to produce free CLA in media supplemented with linoleic acid. Maximum production of CLA (80.14 to 131.63 microg/ml) was observed at 24 h of incubation in broth containing 0.02% of free linoleic acid. No significant (P > 0.05) increases in total CLA levels were observed after 24 h of incubation. The ability of the cultures to produce CLA in skim milk supplemented with 0.02% free linoleic acid also was studied. In this medium, the total amounts of free CLA after 24 h of incubation ranged from 54.31 to 116.53 microg/ml. The use of lactic acid bacteria able to form free CLA in cultured dairy products may have potential health or nutritional benefits. Free CLA in the products likely would be more readily available for absorption from the digestive tract than if it were incorporated into the cells of the starter culture.     

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.     

Screening of some potentially probiotic lactic acid bacteria for their ability to synthesis conjugated linoleic acid

Strains of potentially probiotic lactobacilli, propionibacteria, leuconostoc, lactococcus, enterococcus, and pediococcus, were tested for their ability to convert linoleic acid to conjugated linoleic acid (CLA). Growth and CLA production were followed during incubation for 48 h in reconstituted skim milk containing 0.2% lipolysed sesame oil. Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides subsp. mesenteroides gave the highest CLA production. Also, the effect of lipolysed oil concentration on the growth and CLA production of six strains were studied in medium containing 0.0–1% lipolysed oil. Leuconostoc mesenteroides subsp. mesenteroides and Lac. lactis subsp. lactis biovar diacetylactis gave maximum dienes in medium containing 0.6% and 0.8% lipolysed oil respectively.     

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10³ CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.     

Competition mechanisms of lactic acid bacteria and bifidobacteria: Fermentative metabolism and colonization

Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.     

Optical fibre-based methodology for screening the effect of probiotic bacteria on conjugated linoleic acid (CLA) in curdled milk

A methodology based on optical fibre (OF) detection was developed for screening the potential of CLA production by Lactobacillus casei-01, Lactobacillus acidophilus La-5 and Bifidobacterium lactis B94 in probiotic curdled milk. The OF based methodology was validated by comparison with an analytical method based on gas chromatography–mass spectrometry (GC–MS) and it showed comparable linearity (between 5 and 130 μg), accuracy and detection limits, which ranged from 1.92 to 2.56 μg for CLA methyl ester and oleic acid methyl ester, respectively. Furthermore, the proposed OF based methodology was an advantageous analytical methodology for screening CLA production in probiotic curled milk, due to its compact design and effective cost of analysis.     

Selective enumeration and identification of mixed cultures of Streptococcus thermophilus,Lactobacillus delbrueckii subsp. bulgaricus,L. acidophilus,L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk

This study describes selective plating methodologies for enumeration of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk based on selective antibiotic-free media. Enumeration of S. thermophilus was performed using M17-lactose. MRS-fructose was suitable for enumeration of L. bulgaricus and MRS-maltose for differentiation between L. acidophilus and L. paracasei. The selective enumeration of B. lactis was obtained using MRS-raffinose containing 0.05% LiCl. The bacterial counts obtained using selective methods were equivalent to those under optimum culture conditions at a probability level of 95%. Performance of the methods was verified in fermented milk products where identification of the enumerated species was confirmed by species-specific polymerase chain reaction. This study shows that combination of species-specific polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis has great detection and identification potential for verification of accurate species labelling in fermented milk without prior isolation of the bacteria.     

Influence of bacteria used as adjunct culture and sunflower oil addition on conjugated linoleic acid content in buffalo cheese

The influence of bacteria and sunflower oil addition on conjugated linoleic acid content (CLA) in buffalo cheese was determined. Fresh and short-ripened cheeses were manufactured using the same starter culture and four different adjunct strains previously selected by their CLA production rate. Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum and Streptococcus thermophilus were individually used as adjunct culture. Sunflower oil (SO) was added to obtain a final concentration of 200 μg/ml of linoleic acid. CLA levels in cheese were higher than raw milk, especially after ripening time. SO supplementation increase CLA concentrations in fresh cheeses, except in those manufactured with S. thermophilus as adjunct culture. Both, ripening and SO supplementation showed a positive influence on CLA concentration. Similar texture, acidity and colour were determined in cheeses with or without SO supplementation. Buffalo cheeses manufactured with appropriate adjunct cultures may be a natural source of CLA for human consumption.     

Preparation and properties of probiotic concentrated yoghurt (labneh) fortified with conjugated linoleic acid

Milk of high conjugated linoleic acid (CLA) level (1.25 g per 100 g milk fat) was produced by inclusion of fish oil and rousted soy bean in the ration of Holstein cows as compared to 0.55 g per 100 g milk fat in the milk of animals receiving control diet. Milk of normal (control) and high CLA content (treatment) was spray‐dried. Labneh was made from 20 g L^?1 reconstituted milk using 3 mL per 100 mL yoghurt starter and 2 mL per 100 mL of probiotic cultures of Lactobacillus casei or Lactobacillus acidophilus. The control (C) and high CLA (T) labneh were analysed chemically and microbiologically, and their viscosities were determined during cold storage for 15 days. The fat content of labneh of high CLA was less than that of the control, but the total solids (TS) were unaffected by the CLA level. Labneh made with Lb. acidophilus had lower TS and higher acidity, exopolysaccharides and acetaldehyde contents and viscosity than that made with the use of Lb. casei. Labneh from the different treatments retained high counts of the added probiotic (>10⁸ cfu g^?1) throughout the storage period. The storage period had significant effects on all parameters determined.     

Screening selected strains of probiotic lactic acid bacteria for their ability to produce biogenic amines (histamine and tyramine)

The aim of this study was to investigate the production of biogenic amines (BA), histamine and tyramine by some probiotic lactic acid bacteria (LAB). Fifteen strains representing six LAB species were screened qualitatively by growing them in a decarboxylase medium. Quantitative analysis was carried out by HPLC analysis with direct derivatization of acid extracts. Lactobacillus casei (TISTR 389) and Lactobacillus delbrueckii subsp. bulgaricus (TISTR 895) were found to produce BA. The highest levels of histamine (1820.9 ± 3.5 mg L^?1) and tyramine (5486.99 ± 47.6 mg L^?1) formation were observed for the TISTR 389 strain, while TISTR 895 produced only histamine (459.1 ± 0.63 mg L^?1) in the decarboxylase broth. Biogenic amine potential was not observed for the Lactobacillus acidophilus, Lactobacillus lactis subsp. lactis, Lactococcus lactis subsp. lactis, and Lactobacillus plantarum strains studied. This study confirmed that BA formation is strain dependent and not related to the species. Therefore, careful screening for amino acid decarboxylase activity is recommended before selecting LAB as appropriate starter or probiotic strains in food and dairy industry.     

Formation of conjugated linoleic acid by a Lactobacillus plantarum strain isolated from an artisanal cheese: Evaluation in miniature cheeses

Among 129 lactic acid bacteria previously isolated from raw-milk starter-free cheeses manufactured in Galicia (NW Spain), two strains of Lactobacillus plantarum were definitely recognised as producers of conjugated linoleic acid (CLA). Gas chromatography analysis identified cis-9, trans-11 C18:2 as the predominant CLA isomer formed in MRS broth supplemented with linoleic acid. A centrifugation-based model for the manufacture of miniature cheeses was used to evaluate the formation of CLA by Lb. plantarum L200, the highest producer of CLA in MRS broth. The miniature cheeses made with the addition of the L200 strain showed significantly (P < 0.05) higher contents of cis-9, trans-11 CLA than those of the control cheeses (1.09 versus 0.69 percentage of total fatty acids, respectively). These results suggest that Lb. plantarum L200 strain could be used as an adjunct culture to slightly increase the concentrations of CLA in short-ripened cows' milk cheeses.     

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.     

Lysis of starters in UF cheeses: Behaviour of mesophilic lactococci and thermophilic lactobacilli

The objective of this work was to study the autolytic behaviour of strains of mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic lactic acid bacteria (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus) in UF cheese. Cheeses were made from a UF-retentate (milk concentrated by a factor of 6) of microfiltered milk (0.8 μm pore size membrane) using the following starter systems: (1) single inocula of autolytic strains of L. lactis (US3, AM2 or AM1), non-autolytic strains of L. lactis (AM2-C or CNRZ-144), (2) a co-inocula of strains of Lb. helveticus (ITG-LH1, CNRZ-32 or CNRZ-303), Lb. delbrueckii subsp. lactis (ITG-LL14 or ITG-LL51) with the same strain of S. thermophilus CNRZ-1358. Cell viability was monitored over a 28 day ripening period by enumeration on selective media. Degree of lysis was determined by the measurement of the intracellular marker lactate dehydrogenase (LDH) activity, and also by immunodetection of intracellular proteins with species specific antibodies. In UF cheeses, lysis of autolytic strains of L. lactis was significantly delayed, showing release of intracellular components after 21 days of ripening. No lysis was observed for non-autolytic L. lactis strains or for S. thermophilus. Lysis of thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii), was observed from the start of ripening, but the onset and the level of lysis observed was strain and species dependent.     

Production of conjugated linoleic acid by lactic acid bacteria in milk without any additional substrate

This study was undertaken to evaluate the production and isomeric distribution of conjugated linoleic acid (CLA) in milk fermented with lactic acid bacteria (LAB. A total of 155 cultures of LAB were analysed. Control milk samples had an average CLA content of 0.41 g/100 g of fatty acids (FA), while the lactic cultures produced CLA in the range of 0.43–1.12 g. No major changes in free fatty acids profiles were observed in milk samples fermented with CLA‐producing LAB. The present study demonstrated that LAB can increase levels of CLA in nonsupplemented milk fermented for a short period of 4 h.     

α-Galactosidase and proteolytic activities of selected probiotic and dairy cultures in fermented soymilk

The metabolic activities of Lactobacillus acidophilus (LAFTI^® L10 and La4962) Bifidobacterium (lactis LAFTI^® B94 and longum Bl536), Lactobacillus casei (LAFTI^® L26 and Lc279), Lactobacillus delbrueckii ssp. bulgaricus Lb1466 and Streptococuss thermophilus St1342 were assessed in soymilk. Strains were initially analyzed for α-galactosidase activity and organic acid production in MRS broth at 37 °C. Consequently, soymilk was fermented with each strain and cell growth, production of organic acid, metabolism of oligosaccharides and proteolytic and ACE-inhibitory activities were assessed during 48 h of incubation at 42 °C. All strains exhibited variable α-galactosidase activity, with Bifidobacterium lactis B94 showing the highest activity. The oligosaccharide metabolism depended on α-galactosidase activity. B. lactis B94, S. thermophilus St1342 and L. acidophilus La4962 reduced raffinose substantially by 77.4%, 64.5% and 55.9%, respectively. All strains reached the desired therapeutic level of 10⁸ cfu/ml in soymilk after 48 h at 42 °C. The hydrolysis of protein in soymilk likely depended on strain (P < 0.0001) and time (P < 0.0001). The strains also released bioactive peptides with ACE-inhibitory activities between 17% and 43%.