Effect of Flavourzyme® on Angiotensin‐Converting Enzyme Inhibitory Peptides Formed in Skim Milk and Whey Protein Concentrate during Fermentation by Lactobacillus helveticus

Angiotensin‐converting enzyme inhibitory (ACE‐I) activity as affected by Lactobacillus helveticus strains (881315, 881188, 880474, and 880953), and supplementation with a proteolytic enzyme was studied. Reconstituted skim milk (12% RSM) or whey protein concentrate (4% WPC), with and without Flavourzyme^® (0.14% w/w), were fermented with 4 different L. helveticus strains at 37 °C for 0, 4, 8, and 12 h. Proteolytic and in vitro ACE‐I activities, and growth were significantly affected (P < 0.05) by strains, media, and with enzyme supplementation. RSM supported higher growth and produced higher proteolysis and ACE‐I compared to WPC without enzyme supplementation. The strains L. helveticus 881315 and 881188 were able to increase ACE‐I to >80% after 8 h of fermentation when combined with Flavourzyme^® in RSM compared to the same strains without enzyme supplementation. Supplementation of media by Flavourzyme^® was beneficial in increasing ACE‐I peptides in both media. The best media to release more ACE‐I peptides was RSM with enzyme supplementation. The L. helveticus 881315 outperformed all strains as indicated by highest proteolytic and ACE‐I activities.     

Effects of fermentation conditions on the potential anti‐hypertensive peptides released from yogurt fermented by Lactobacillus helveticus and Flavourzyme®

This study investigates the effects of fermentation conditions on the production of angiotensin‐converting enzyme inhibitory (ACE‐I) peptides in yogurt by Lactobacillus helveticus 881315 (L. helveticus) in the presence or absence of Flavourzyme^®, which is derived from a mould, Aspergillus oryzae and used for protein hydrolysis in various industrial applications. Optimal conditions for peptides with the highest ACE‐I activity were 4% (v/w) inoculum size for 8 h without Flavourzyme^® supplementation, and 1% inoculum size for 12 h when combined with Flavourzyme^®. The yogurt fermented by L. helveticus resulted in IC₅₀ values (concentration of inhibitor required to inhibit 50% of ACE activity under the assayed conditions) of 1.47 ± 0.04 and 16.91 ± 0.25 mg mL^?1 with and without Flavourzyme^® respectively. Seven fractions of ACE‐I peptides from the yogurt incorporated with L. helveticus and Flavourzyme^® were separated using the preparative high‐performance liquid chromatography. Fraction (F3) showed the highest ACE‐I activity with an IC₅₀ of 35.75 ± 5.48 μg mL^?1. This study indicates that yogurt may be a valuable source of ACE‐I peptides, which may explain the outcomes observed in the experimental and clinical studies and foresee the application of fermented milk proteins into functional foods or dietary supplements.     

Probiotic monocultures in fermented goat milk beverages – sensory quality of final product

The aim of our study was to conduct a selection of the monocultures capable of providing the most attractive sensory features of the final product. Four fermented goat's milk beverages were produced with probiotic monocultures containing Lactobacillus (Lb. acidophilus La‐5, Lb. rhamnosus K3 and Lb. plantarum O20) and Bifidobacterium (Bif. animalis subsp. lactisBB‐12). A sensory analysis and microbiological assessment of fermented goat's milk beverages were made at the beginning of the study and after 3, 7, 10 and 14 days of refrigerated storage (5 ± 1 °C). We found that samples including monocultures Lb. plantarum O20 and Bif. animalis subsp. lactisBB‐12 were differentiated from other goat's milk beverages.     

Performance of different microbial cultures in potentially probiotic and prebiotic yoghurts from cow and goat milks

Lactobacillus acidophilus or Bifidobacterium animalis subsp. lactis Bb‐12 and green banana pulp were used in order to obtain potentially probiotic and prebiotic yoghurts, which were compared over a 45‐day storage period. Goat milk yoghurts demonstrated probiotic effects up to 45 days of storage. Cow milk yoghurts produced with B. animalis subsp. lactis Bb‐12 showed a probiotic effect reduction during the storage period (1.74 log CFU/g). The type of milk affected the yoghurts’ chemical and physicochemical properties. Sensory acceptance was also affected, where cow milk yoghurts were better accepted than goat milk ones.     

Cream cheese as a symbiotic food carrier using Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and inulin

The stability of cream cheeses as a symbiotic food carrier, through supplementation with different concentrations of probiotic bacteria Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and the prebiotic ingredient inulin was investigated. Physicochemical parameters, pH values, total solids, fat and protein levels and the viable counts of the starter lactic culture Streptococcus thermophilus and probiotic cultures, were carried out at 1, 15, 30 and 45 days of refrigerated storage (8 ± 0.5 °C). Different physicochemical characteristics were observed in all formulations. S. thermophilus showed good viability in all the trials (6.66–9.38 log cfu/g), whereas B. animalis remained above 6 log cfu/g in all the trials during the period evaluated. However, L. acidophilus showed an accentuated decline, registering values of 3.1 log cfu/g at the end of the period studied. The results suggested that cream cheese was an adequate food matrix for supplementation with probiotic bacteria, in particular B. animalis, and the prebiotic ingredient, showing potential as a symbiotic food.     

Protein hydrolysate from tilapia frame: antioxidant and angiotensin I converting enzyme inhibitor properties

The effect of varying the aminopeptidase concentration and hydrolysis time in the production of a protein hydrolysate from tilapia frame (TFPH) was evaluated in terms of the obtained antioxidant and angiotensin I converting enzyme (ACE) inhibitor activities. Minced tilapia frame was subjected to hydrolysis using three concentrations Flavourzyme^® 1000 L [Brenntag Ingredients (Thailand) Company Limited (Public), Bangkok, Thailand] [0%, 1% and 2% (w/w)] and two hydrolysis times (0 and 1 h). The enzyme concentration and hydrolysis time both significantly affected the antioxidant and ACE inhibitor properties. The use of 2% (w/w) Flavourzyme^® 1000 L for 1 h yielded the highest levels of 2,2‐diphenyl‐1‐picrylhydrazil free radical scavenging (90.4%), metal chelating (91.8%), thiobarbituric acid activity ratio (81.9%), and ACE inhibition (83.8%). In addition, this TFPH contained a higher net amount of amino acids and a larger peptide molecular weight distribution compared to the unhydrolysed tilapia frame supernatant. Thus, TFPH may be a suitable supplement to improve the functionality of food products.     

Developing a potential prebiotic of yogurt: growth of Bifidobacterium and yogurt cultures with addition of glycomacropeptide hydrolysate

This study is aimed at investigating the feasibility of casein glycomacropeptide (GMP) hydrolysates as potential prebiotics in yogurt. The growth performance of Bifidobacterium animalis subsp. lactis (Bb12) was determined in the presence of GMP hydrolysates produced by the action of proteolytic enzymes (trypsin and papain) with various degrees of hydrolysis. The growth‐promoting effect of GHP on Lactobacillus bulgaricus and Streptococcu thermophilus were also evaluated. Results showed that the optimum hydrolysis time of GMP with trypsin or papain for Bb12 growth promotion was 2 and 0.5 h, respectively. Compared with GMP and its trypsin hydrolysate, the GMP hydrolysate produced with papain (GHP) was the best for Bb12 growth, which obtained the highest viable count (9.3 log cfu mL^?1) and the lowest pH of 4.69. The obtained GHP significantly improved the growth of S. thermophilus (P < 0.05), while it had little effect on the growth of L. bulgaricus (P > 0.05)., The viable count of Bb12 of the yogurt obtained with the addition of 1.5% GHP was about four times higher than that of the control without GHP addition. The growth‐promoting effect of GHP might be related to its high content of Glu, Leu and Ala while had no direct relationship with sialic acid content.     

Probiotic Strains as Starter Cultures Improve Angiotensin-converting Enzyme Inhibitory Activity in Soy Yogurt

Suitability of soy yogurt as a system for delivering probiotics and other bioactive compounds was assessed by fermenting soy milk using starter culture containing Lactobacillus delbrueckii ssp. bulgaricus Lb1466, Streptococcus thermophilus St1342, and probiotic organisms (Lactobacillus acidophilus LAFTI® L10, Bifidobacterium lactis LAFTI® B94, and Lactobacillus paracasei LAFTI® L26). Fermentations were terminated at different pH of 4.50, 4.55, and 4.60 and metabolic patterns of cultures (viability, proteolytic activity, organic acids production, angiotensin‐converting enzyme (ACE) inhibitory activity) were investigated during 28 d of storage at 4 °C. The presence of probiotics enhanced the growth of L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St134 in soy yogurt in comparison to the control produced by sole yogurt culture. In general, different termination pH had no effect (P > 0.05) on the viability of probiotic organisms that maintained good viability in soy yogurt during cold storage. Higher levels of essential growth factors in the form of peptides and amino acids in soy yogurts may have promoted the growth of L. acidophilus LAFTI® L10, B. lactis LAFTI® B94, and L. paracasei LAFTI® L26. The use of probiotic strains as a part of starter culture in soy yogurt resulted in a substantial increase in in vitro ACE inhibitory activity compared with the control produced by yogurt culture only. This improvement of ACE inhibition in soy yogurt is partly due to higher proteolytic activity of probiotics.     

Effects of enzymatic hydrolysis treatments on the physicochemical properties of beef bone extract using endo- and exoproteases

This study reported the effects of enzymatic hydrolysis treatments on the physiochemical properties of beef bone extract using endo- and exoproteases. Each enzyme hydrolysis kinetics were studied using Michaelis–Menten model, and the ideal E/S ratio obtained for Protamex^® (P), bromelain (B) and Flavourzyme^® (F) was found to be 1.10%, 1.60% and 4.70% w/w, respectively. Seven hydrolysates were produced from single (P, B, F), simultaneous (P + F, B + F) and sequential (P > F, B > F) treatments, where bone extract hydrolysed by Flavourzyme^® exhibited highest DH and proportion of low molecular weight (Mw) peptides (<5000 Da) in single treatment. When Flavourzyme^® was used with Protamex^® or bromelain in simultaneous or sequential treatments, no significant differences in Mw distribution, exposed SH content, SS content and viscosity were evident compared with Flavourzyme^® only. This indicated that without the addition of other enzymes, Flavourzyme^® was capable of increasing the proportion of low Mw peptides and reduce viscosity.     

Influence of adjunct cultures on angiotensin‐converting enzyme (ACE)‐inhibitory activity,organic acid content and peptide profile of kefir

The angiotensin‐converting enzyme (ACE)‐inhibitory activities, peptide profiles and organic acid contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were determined. All the kefir samples showed almost similar peptide profiles as detected by RP‐HPLC, but quantitative differences were observed during storage. The ACE‐inhibitory activities of different lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage, there was a significant increase in ACE‐inhibitory activity of the sample fermented with Lactobacillus helveticus. However, a kefir sample containing Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE‐inhibitory activity (92.23%) compared to the other samples.     

Angiotensin‐I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates

BACKGROUND: Enzymatic proteolysis of food proteins is used to produce peptide fractions with the potential to act as physiological modulators. Fractionation of these proteins by ultrafiltration results in fractions rich in small peptides with the potential to act as functional food ingredients. The present study investigated the angiotensin‐I converting enzyme (ACE‐I) inhibitory and antioxidant activities for hydrolysates produced by hydrolyzing Vigna unguiculata protein extract as well as ultrafiltered peptide fractions from these hydrolysates. RESULTS: Alcalase^®, Flavourzyme^® and pepsin–pancreatin were used to produce extensively hydrolyzed V. unguiculata protein extract. Degree of hydrolysis (DH) differed between the enzymatic systems and ranged from 35.7% to 58.8%. Fractionation increased in vitro biological activities in the peptide fractions, with IC₅₀ (hydrolysate concentration in µg protein mL^?1 required to produce 50% ACE inhibition) value ranges of 24.3–123 (Alcalase hydrolysate, AH), 0.04–170.6 (Flavourzyme hydrolysate; FH) and 44.7–112 (pepsin–pancreatin hydrolysate, PPH) µg mL^?1, and TEAC (Trolox equivalent antioxidant coefficient) value ranges of 303.2–1457 (AH), 357.4–10 211 (FH) and 267.1–2830.4 (PPH) mmol L^?1 mg^?1 protein. CONCLUSION: The results indicate the possibility of obtaining bioactive peptides from V. unguiculata proteins by means of a controlled protein hydrolysis using Alcalase^®, Flavourzyme^® and pepsin–pancreatin. The V. unguiculata protein hydrolysates and their corresponding ultrafiltered peptide fractions might be utilized for physiologically functional foods with antihypertensive and antioxidant activities. Copyright © 2010 Society of Chemical Industry     

Colloidal aggregation induced by the reduction in pH and the synthesis of new molecular structures during the milk fermentation process

The use of different inocula in the milk‐to‐yoghurt transformation influences the kinetics of the fermentation process. The aim of this work was to study the kinetics induced by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lb–St). The milk‐to‐yoghurt transformation showed no delay in the production of lactic acid for yoghurt produced using Lactobacillus johnsonii with S. thermophilus (La1–St) or Lactobacillus casei with S. thermophilus (Lc1–St); the delays were 20–70 min and 40–80 min, respectively. The absence of delay was 1.0/min (Lb–St) as compared with 0.015/min (La1–St) and 0.7/min (Lc1–St). The kinetics was fitted using second‐order reaction.     

Effect of temperature on growth and metabolism of probiotic bacteria in milk

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT) treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 °C and the samples were analysed for pH, log cfu mL⁻¹, volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell numbers above 6.5 log cfu mL⁻¹ after 48 h fermentation at 30, 37 and 45 °C. The probiotic strains produced different amounts of metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

A comparative study of physico‐chemical properties and antioxidant activity of freeze‐dried mung bean (Vigna radiata) and adzuki bean (Vigna angularis) sprout hydrolysate powders

Mung bean (MB) and adzuki bean (AZB) sprouts were hydrolysed with Flavourzyme^® at four different concentrations for 6 h. Nongerminated beans subjected to each enzyme concentrations were set as the control. For both bean sprouts, the highest amounts of free amino group, total free amino acids and total phenolic content (TPC) were obtained with 7% (w/w) Flavourzyme^®. Each bean sprout hydrolysate was subjected to freeze‐drying in absence and presence of 5% maltodextrin (w/v). The addition of maltodextrin resulted in a decrease in the free amino group, TPC, surface hydrophobicity and hygroscopicity in resulting freeze‐dried powders. Gallic acid, p‐coumaric acid and vitexin were identified in all freeze‐dried powders, while catechin and rutin were detected only in freeze‐dried AZB hydrolysate powder. Freeze‐dried AZB hydrolysate powder contained higher antioxidant activities. DPPH radical scavenging activity of all samples measured using electron spin resonance spectrometry was higher than that obtained by the colorimetric method.     

Effect of different matrices on probiotic resistance to in vitro simulated gastrointestinal conditions

In this study, we evaluated the effect of different matrices (MRS, milk and milk with inulin) on the tolerance of probiotic strains (Lactobacillus acidophilus La‐5 and Bifidobacterium animalis subsp. lactis BB‐12) to simulated conditions similar to those found in the gastrointestinal tract. Both probiotic strains demonstrated a significantly lower viability after exposure to in vitro gastric and intestinal conditions, and B. animalis subsp. lactis BB‐12 showed higher survival during the test compared to L. acidophilus La‐5 in all tested matrices. Milk and inulin protected probiotics from in vitro gastrointestinal stress. These results suggest that it is critical to formulate the food matrix to be used as probiotic carrier.     

Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris

This research focuses on green production of bioactive proteins and hydrolysates from Nitzschia. A comparison of antioxidant activities was established between protein extracts and hydrolysates from Nitzschia and two other well‐known microalgae, chlorella and spirulina. Protein hydrolysates from these microalgae were produced using Alcalase^®, Flavourzyme^® and Trypsin. The hydrolysis process enhanced the antioxidant activities in general, especially those obtained using Alcalase^®. Nitzschia showed the highest (P < 0.05) total phenolic content/reducing capacity (2.4 ± 0.02 mg GAE/100 g) after 90 min of hydrolysis with Alcalase^®. The ABTS [2,2′‐Azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid)] radical scavenging activity (66.77 ± 0.00%) was highest (P < 0.05) after 120 min of hydrolysis, but DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl radical) was low (29.59 ± 0.02%). A correlation between ABTS activity and total phenolic contents was the highest (P < 0.05) for protein hydrolysates from all three organisms using Alcalase^®, but superoxide anion radical scavenging activity was intermediate for Nitzschia. Therefore, Nitzschia protein hydrolysates have the potential to be used as antioxidants.     

Interaction between lactic acid bacteria and food‐borne pathogens on putrescine production in ornithine‐enriched broth

Interactions of mixed cultures [lactic acid bacteria (LAB) and food‐borne pathogens (FBP)] on putrescine (PUT) as well as other biogenic amines (BAs) production were investigated in ornithine‐enriched broth. Significant differences in BAs production were found among the bacterial strains (P < 0.05). Conversion of ornithine into PUT by Salmonella Paratyphi A and Aeromonas hydrophila as well as Listeria monocytogenes and Staphylococcus aureus was high (>75 mg L^?1), whereas other bacterial strains yielded below 50 mg L^?1 of PUT. LAB strains resulted in significant reduction in PUT by Pseudomonas aeruginosa and Enterobacteriaceae, except for Escherichia coli, which was stimulated more than two‐fold PUT in the presence of Lactococcus lactis subsp. lactis. Lactobacillus plantarum had generally inhibition effect on histamine (HIS) and tyramine production by FBP, whereas Lc. lactic subsp. lactic slightly stimulated HIS by E. coli and A. hydrophila. Streptococcus thermophilus resulted in 1.5‐fold higher HIS formation by bacteria (10 mg L^?1). Consequently, the interaction between LAB and specific FBP might result in significant inhibition of amine accumulation, if the correct LAB strains are used.     

Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Analysis of volatile compounds produced by different species of lactobacilli in rye sourdough using multiple headspace extraction

Profiles of volatile organic compound (VOC) produced by nine individual lactic acid bacteria (LAB) during rye sourdough fermentation were compared by automated SPME and GC/MS‐Tof. The dough samples were inoculated with individual strains, placed inside the headspace vials and incubated during next 24 h. The production or loss of VOC‐s was followed by adsorbing volatiles onto 85‐m Car/PDMS fibre in every 4 h. Volatile profiles differed among LAB species and divided LAB into two main groups – hetero‐ and homofermentative. Hetrofermentative LAB (Lactobacillus brevis; Leuconostoc citreum; Lactobacillus vaginalis, Lactobacillus panis) showed high production of acetic acid, CO₂, ethanol, ethylacetate, producing also hexyl acetate, ethyl hexanoate and isopentyl acetate. Whereas homofermentative LAB species (Lactobacillus helveticus; Lactobacillus casei; Lactobacillus sakei; Lactobacillus curvatus) produced a considerable amount of 2,3‐butanedione. Production of l ‐leucine methyl ester was unique for Lb. sakei, Lb. casei and Lb. curvatus strains. Lb. helveticus was the only LAB that produced benzaldehyde.