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.     

Physiochemical Properties,Microstructure, and Probiotic Survivability of Nonfat Goats’ Milk Yogurt Using Heat‐Treated Whey Protein Concentrate as Fat Replacer

There is a market demand for nonfat fermented goats’ milk products. A nonfat goats’ milk yogurt containing probiotics (Lactobacillus acidophilus, and Bifidobacterium spp.) was developed using heat‐treated whey protein concentrate (HWPC) as a fat replacer and pectin as a thickening agent. Yogurts containing untreated whey protein concentrate (WPC) and pectin, and the one with only pectin were also prepared. Skim cows’ milk yogurt with pectin was also made as a control. The yogurts were analyzed for chemical composition, water holding capacity (syneresis), microstructure, changes in pH and viscosity, mold, yeast and coliform counts, and probiotic survivability during storage at 4 °C for 10 wk. The results showed that the nonfat goats’ milk yogurt made with 1.2% HWPC (WPC solution heated at 85 °C for 30 min at pH 8.5) and 0.35% pectin had significantly higher viscosity (P < 0.01) than any of the other yogurts and lower syneresis than the goats’ yogurt with only pectin (P < 0.01). Viscosity and pH of all the yogurt samples did not change much throughout storage. Bifidobacterium spp. remained stable and was above 10⁶CFU g^‐1 during the 10‐wk storage. However, the population of Lactobacillus acidophilus dropped to below 10⁶CFU g^‐1 after 2 wk of storage. Microstructure analysis of the nonfat goats’ milk yogurt by scanning electron microscopy revealed that HWPC interacted with casein micelles to form a relatively compact network in the yogurt gel. The results indicated that HWPC could be used as a fat replacer for improving the consistency of nonfat goats’ milk yogurt and other similar products.     

Effect of commercial tannin and a pornunça (Manihot spp.) silage‐based diet on the fatty acid profile of Saanen goats’ milk

The aim of this study was to evaluate the performance, milk composition, blood and urinary biochemical parameters and milk fatty acid profile of Saanen goats fed pornunça silage‐based diets containing different levels of tannin (11, 28, 36 and 44 g/kg dry matter). Intake of feed, milk production, milk composition and biochemical parameters were affected (P < 0.05). The content of 10:0, 12:0 and 14:0 fatty acids decreased, while those of 18:0, cis‐9 18:1, trans‐11 18:1, cis‐9, trans‐11 18:2 and cis‐9, cis‐12, cis‐15 18:3 increased as more tannin was added (P < 0.05). The ratio of unsaturated to saturated fatty acids increased, while the thrombogenicity and atherogenicity indices decreased as tannin was increased (P < 0.05). The inclusion of up to 28 g/kg of tannin was the most appropriate level to improve the fatty acid profiles of goats’ milk and prevent further falls in dry matter intake and milk production.     

Comparison of volatile compounds in yoghurts made from cows’, buffaloes’, ewes’ and goats’ milks

The composition of the volatile fraction of yoghurts made from cows’, buffaloes’, ewes’ and goats’ milks was investigated during the 28 days of storage at 4 °C using solid‐phase microextraction technique (SPME) and gas chromatography coupled to mass spectrometry analysis (GC/MS). A total of 34 volatile compounds were identified in yoghurts during their storage at 4 °C, including aldehydes, ketones, alcohols, esters, acids, terpenes, hydrocarbons and sulphur compounds. In this study, acetaldehyde, diacetyl and acetoin, considered as the major compounds of yoghurt, were detected in all yoghurts.     

Calcium–Alginate–Inulin Microbeads as Carriers for Aqueous Carqueja Extract

Carqueja (Pterospartum tridentatum) is an endemic species and various bioactive compounds have been identified in its aqueous extract. The aim of this study was to protect the natural antioxidants from the aqueous extract of carqueja by encapsulation in Ca–alginate microbeads and Ca–alginate microbeads containing 10% and 20% (w/v) of inulin. The microbeads produced by electrostatic extrusion technique had an average diameter from 625 μm to 830 μm depending on the portion of inulin. The sphericity factor of the hydrogel microbeads had values between 0.014 and 0.026, while freeze dried microbeads had irregular shape, especially those with no excipient. The reduction in microbeads size after freeze drying process (expressed as shrinkage factor) ranged from 0.338 (alginate microbeads with 20% (w/v) of inulin) to 0.523 (plain alginate microbeads). The expressed radical scavenging activity against ABTS and DPPH radicals was found to be between 30% and 40% for encapsulated extract, while the fresh extract showed around 47% and 57% of radical scavenging activity for ABTS and DPPH radicals, respectively. The correlation between antioxidant activity and the total phenolic content were found to be positive (in both assay methods, DPPH and ABTS), which indicate that the addition of inulin didn't have influence on antioxidant activity. The presence of inulin reduced stiffness of the hydrogel, and protected bead structure from collapse upon freeze‐drying. Alginate–inulin beads are envisaged to be used for delivery of aqueous P. tridentatum extract in functional food products.     

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.     

Preparation of Acid‐Resistant Microcapsules with Shell‐Matrix Structure to Enhance Stability of Streptococcus Thermophilus IFFI 6038

Microencapsulation is an effective technology used to protect probiotics against harsh conditions. Extrusion is a commonly used microencapsulation method utilized to prepare probiotics microcapsules that is regarded as economical and simple to operate. This research aims to prepare acid‐resistant probiotic microcapsules with high viability after freeze‐drying and optimized storage stability. Streptococcus thermophilus IFFI 6038 (IFFI 6038) cells were mixed with trehalose and alginate to fabricate microcapsules using extrusion. These capsules were subsequently coated with chitosan to obtain chitosan‐trehalose‐alginate microcapsules with shell‐matrix structure. Chitosan‐alginate microcapsules (without trehalose) were also prepared using the same method. The characteristics of the microcapsules were observed by measuring the freeze‐dried viability, acid resistance, and long‐term storage stability of the cells. The viable count of IFFI 6038 in the chitosan‐trehalose‐alginate microcapsules was 8.34 ± 0.30 log CFU g^?1 after freeze‐drying (lyophilization), which was nearly 1 log units g^?1 greater than the chitosan‐alginate microcapsules. The viability of IFFI 6038 in the chitosan‐trehalose‐alginate microcapsules was 6.45 ± 0.09 log CFU g^?1 after 120 min of treatment in simulated gastric juices, while the chitosan‐alginate microcapsules only measured 4.82 ± 0.22 log CFU g^?1. The results of the long‐term storage stability assay indicated that the viability of IFFI 6038 in chitosan‐trehalose‐alginate microcapsules was higher than in chitosan‐alginate microcapsules after storage at 25 °C. Trehalose played an important role in the stability of IFFI 6038 during storage. The novel shell‐matrix chitosan‐trehalose‐alginate microcapsules showed optimal stability and acid resistance, demonstrating their potential as a delivery vehicle to transport probiotics.     

Encapsulation of probiotic Lactobacillus acidophilus by ionic gelation with electrostatic extrusion for enhancement of survival under simulated gastric conditions and during refrigerated storage

The probiotic Lactobacillus acidophilus was encapsulated in biodegradable and biocompatible capsules prepared by ionic gelation between phytic acid (PA) and chitosan (CS) with an electrostatic extrusion method. Calcium carbonate (CaCO₃) and starch were used as co‐encapsulants for improvement of capsule stability. Capsules were characterised and evaluated for survival of encapsulated L. acidophilus cells in simulated gastric fluid (SGF) and during refrigerated storage. Loading capacity values of PA‐CS capsules, PA‐CS‐starch capsules and PA‐CS‐CaCO₃ capsules were 8.20, 8.12 and 7.81 log CFU g^?1 of wet capsule, respectively. Capsules showed particle sizes of 1.3–1.5 mm and a uniform spherical shape. PA‐CS‐CaCO₃ capsules were the most stable vehicles for the protection of probiotic cells against acidic damage, particularly at pH 1.5 and pH 2. L. acidophilus cells from PA‐CS‐CaCO₃ capsules showed only a 0.64 log CFU reduction in numbers after 2 h in pH 1.5 SGF conditions. The numbers of L. acidophilus encapsulated in PA‐CS‐CaCO₃ capsules were decreased by only 0.69 log CFU g^?1, while PA‐CS capsules and PA‐CS‐starch capsule numbers were reduced by more than 1.45 log CFU g^?1 after 4 weeks at 4 °C. Addition of calcium carbonate to PA‐CS capsules provided protection against acid injury via antacid and buffering effects for encapsulation of L. acidophilus.     

The effect of co-encapsulation of Lactobacillus rhamnosus GG ATCC 53103 with inulin on alginate/chitosan matrix: the viability in fermented soy blend and simulated digestive system

This research verified the ability of Lactobacillus rhamnosus encapsulated with inulin to tolerate the simulated digestive system and their viability in a soy blend. Probiotic encapsulated in alginate-chitosan matrix without inulin presented a better encapsulation efficiency (80.92%) than encapsulation with inulin (57.39%). On the 28th day, the count of probiotics decreased by 3.42 and 1.99 logarithmic cycles of free and encapsulated cells without inulin, respectively. In contrast, the microorganisms encapsulated with inulin showed an increase of 1.26 logs CFU g⁻¹. During gastrointestinal simulation, cell counts decreased by 0.78, 1.55 and 1.95 CFU g⁻¹ logs for encapsulated cells without inulin, free and encapsulated with inulin, respectively. Sensory panellists liked the fermented soy blend with encapsulated lactobacilli, and this result shows the possibility to create new probiotic foods of plant origin. Therefore, the alginate/chitosan matrix can be considered adequate for the encapsulation of L. rhamnosus. The inulin reduces the encapsulation efficiency and increases the cell loss in gastrointestinal simulation. Considering cellular losses, the best option for preparing a fermented soy blend is to use L. rhamnosus encapsulated without inulin.     

Characterisation of the vegetable rennets used for ‘Torta del Casar’ cheesemaking by a protein profile method

The purpose of this work was to develop a cheap, easy and fast procedure, based on protein analysis, that can be used in the characterisation of coagulants from thistle species, which can be used in Torta del Casar cheesemaking. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis showed high efficiency in the identification of Cynara cardunculus from other thistle species and in discriminating between the technological properties, milk‐clotting and casein degradation activities of the vegetable rennets used in this study. This technique allowed for the grouping of aqueous extracts from the flowers of C. cardunculus into five different groups, which exhibited variable ability to degrade milk caseins.     

Application of chitosan–alginate microspheres for the sustained release of bacteriophage in simulated gastrointestinal conditions

This study was designed to evaluate the acid stability, release property and antimicrobial efficacy of Escherichia coli O157:H7 bacteriophages encapsulated in chitosan–alginate microspheres under the simulated gastrointestinal conditions. The bacteriophages belonging to Myoviridae family were stable at the pH above 4 in trypticase soy broth. The chitosan–alginate microspheres exhibited protective effect on the viability of bacteriophages in the simulated gastric conditions at pH 2.0 and pH 2.5, showing 4.8 and 5.6 log PFU mL^‐1, respectively, after 1 h of incubation at 37 °C. The release per cent of bacteriophages from microspheres gradually increased up to 65% in the simulated intestinal condition (pH 7.5) at 37 °C for 6 h. The lytic efficacy of chitosan‐ and alginate‐encapsulated bacteriophages against E. coli O157:H7 was significantly maintained in the simulated intestinal conditions to 10 h of incubation (1.3 log reduction). The results suggest that the chitosan–alginate microspheres can be used as a reliable delivery system for bacteriophages.     

The effects of ratio of cow's milk to soymilk,probiotic strain and fruit concentrate on qualitative aspects of probiotic flavoured fermented drinks

The effects of proportion of cow's milk to soymilk (100:0, 75:25, 50:50, 25:75, 0:100), probiotic bacteria (Lactobacillus acidophilus LA‐5 or Lactobacillus casei L‐01) and natural fruit concentrates (strawberry, apricot, peach and pear) on quality characteristics of soy‐based probiotic drink were investigated. The parameters were analysed at the end of fermentation and during 21 days of storage at 5 ^°C. The highest viability was observed when the equal proportion of cow's milk and soymilk and L. casei was used (50:50‐CY). During chilled storage, the flavouring apricot had the highest stimulatory effect on the survival of L. casei in 50:50 treatment. In general, the treatment 50:50‐CY was realised as the best one overall.     

Effects of Mentha longifolia L. essential oil and Lactobacillus casei on the organoleptic properties and on the growth of Staphylococcus aureus and Listeria monocytogenes during manufacturing,ripening and storage of Iranian white‐brined cheese

This research evaluated the effects of Mentha longifolia L. essential oil (EO) in concentrations 0, 50, 150 and 300 ppm and Lactobacillus casei (10⁸–10⁹ CFU/mL) on the growth of Staphylococcus aureus and Listeria monocytogenes during the manufacturing, ripening and storage of Iranian white‐brined cheese. The growth of the two pathogens was significantly reduced (P < 0.01) by both EO concentrations ≥50 ppm and probiotic and their combination in the standard manufacturing and storage process conditions of the cheese. Furthermore, the treatment containing 150 ppm of this EO combined with probiotic had a favourable inhibitory effect on the growth of two pathogenic micro‐organisms and also was the most appropriate treatment in sensory assessment. The synergistic effects of the above‐mentioned concentration level between the essential oil and probiotic were significant compared to other treatments, including essential oil and probiotic only. Thus, a lower concentration of this EO can be used when it is combined with this probiotic.     

Effect of long‐chain inulin on the texture profile and survival of Lactobacillus paracasei ssp. paracasei in set yoghurts during refrigerated storage

The effects of adding inulin at 20 g/L as a fat replacer and probiotic bacteria on the physicochemical and textural characteristics of yoghurt were studied. The ability of long‐chain inulin to improve the probiotic (Lactobacillus paracasei ssp. paracasei) bacteria viability in yoghurt was investigated. The addition of inulin made the texture (firmness, cohesiveness, adhesiveness and gumminess) of skimmed yoghurt similar to that of whole yoghurt, demonstrating the role of inulin as a fat replacer. However inulin increased syneresis and did not influence the viability of probiotic bacteria in the yoghurts. The addition of probiotic bacteria in yoghurts improved syneresis and increased firmness and gumminess.     

Study of the effects of spray‐drying on the functionality of probiotic lactobacilli

Three probiotic lactobacilli strains were spray‐dried in 20% (w/v) skim milk and submitted to a simulated gastrointestinal digestion. Fresh or spray‐dried cultures were administered to mice for 5 and 10 days, and Immunoglobulin A (IgA)‐producing cells were enumerated in the small intestine by immunohistochemistry. Spray‐drying significantly enhanced the resistance of Lactobacillus paracasei A13 and Lactobacillus casei Nad to a simulated gastrointestinal digestion (0.96 and 1.95 log orders, respectively), compared with fresh cultures. Also, a significant higher number of IgA‐producing cells were induced by spray‐dried cultures compared with fresh cultures. Spray‐drying is a suitable, but strain‐dependent, technological process for the development of probiotic cultures in skim milk with increased functionality.     

An Improved Method of Microencapsulation of Probiotic Bacteria for Their Stability in Acidic and Bile Conditions during Storage

ABSTRACT: The purpose of this study was to develop a method for applying an extra coating of palm oil and poly‐L‐lysine (POPL) to alginate (ALG) microcapsules to enhance the survival of probiotic bacteria. Eight strains of probiotic bacteria including Lactobacillus rhamnosus, Bifidobacterium longum, L. salivarius, L. plantarum, L. acidophilus, L. paracasei, B. lactis type Bl‐O4, and B. lactis type Bi‐07 were encapsulated using alginate alone or alginate with POPL. Electron microscopy was used to measure the size of the microcapsules and to determine their surface texture. To assess if the addition of POPL improved the viability of probiotic bacteria in acidic conditions, both ALG and POPL microcapsules were inoculated into pH 2.0 MRS broths and their viability was assessed over a 2‐h incubation period. Two bile salts including oxgall bile salt and taurocholic acid were used to test the bile tolerance of probiotic bacteria entrapped in ALG and POPL microcapsules. To assess the porosity and the ability of the microcapsule to hold small molecules in an aqueous environment a water‐soluble fluorescent dye, 6‐carboxyflourescin (6 FAM), was encapsulated and its release was monitored using a UV spectrophotometer. The results indicated that coating the microcapsules with POPL increased the overall size of the capsules by an average of 3 μm ± 0.67. However, microcapsules with added POPL had a much smoother surface texture when examined under an electron microscope. The results also indicated that the addition of POPL to microcapsules improved the average viability of probiotic bacteria by > 1 log CFU/mL when compared to ALG microcapsules at 2 h of exposure to acidic conditions. However, similar plate counts were observed between ALG and POPL microcapsules when exposed to bile salts. This suggests that an extra coating of POPL could be readily broken down by bile salts that are commonly found in the lower gastrointestinal tract (GIT). Upon testing the porosity of the microcapsules, findings suggest that POPL microcapsules were less porous and hold 52.2% more fluorescent dye over a 6‐wk storage period.     

Detection and quantification of Escherichia coli and Pseudomonas aeruginosa in cow milk by near‐infrared spectroscopy

This work investigated the potential of NIR technology to be applied in the dairy industry for the detection of micro‐organisms. To this end, two types of cow milk samples were studied, one in which only bacterial biomass was considered and the other in which bacteria were cultured and grown in milk for 24 h. The study was carried out using two micro‐organisms Escherichia coli and Pseudomonas aeruginosa. Both types of samples with different counts of both micro‐organisms were analysed by a NIR analyser in the range 10 000–4000 cm^?1 based on transmittance measurements. Multivariate models indicated that a better discrimination between micro‐organisms was attained in those milk samples in which micro‐organisms have been grown.     

Optimisation of the formulation for barley–milk composite‐based fermented drink

The effect of barley flour concentration, Lactobacillus plantarum NCDC344 (Lp344) and co‐culture (Streptococcus thermophilus 20) inoculum levels on the sensory quality, Lp344 count, β‐glucan content and viscosity of barley–milk composite‐based fermented drink was investigated. A central composite rotatable design of response surface methodology was used for optimisation of the formulation. Of the three formulation variables, barley flour concentration was found to be the most critical as it significantly affected overall acceptability, Lp344 count and β‐glucan content (P < 0.01). The optimised drink rated 7.80 on a 9‐point hedonic scale, and contained 8.59 log cfu/mL of Lp344 cells and 0.144 g/100 g of β‐glucan.     

Evaluation of physiochemical,textural, microbiological and sensory characteristics in set yogurt reinforced by microencapsulated Bifidobacterium bifidum F‐35

The objectives of this study were to encapsulate the Bifidobacterium bifidum F‐35 into whey protein for the production of one‐layer microcapsules, and then the microcapsules were covered by sodium alginate to produce double‐layer microcapsules for examining the effectiveness of microcapsules in set yogurt. The reinforced treatment by double layer exhibited a significant increase (P < 0.05) in B. bifidum F‐35 count more than the treatments of free cells and one‐layer microcapsules. Microcapsules of double layer in yogurt led to record a value of titratable acidity that was 1.51 in comparison with the treatments of one layer and free cells that were 1.65 and 1.73, respectively. The hardness values were recorded as 206.88 at the treatment of double layer and 130.31 at the treatment of one layer after 7 days of storage. Microencapsulation of double layer caused a slight bitterness and creamy texture in yogurt, whereas the samples of free cells were described to have sour and bad texture.     

Effect of probiotic co-cultures on physico-chemical and biochemical properties of small ruminants’ fermented milk

Small ruminants' fermented probiotic milk is an alternative to fermented cows' milk, especially because of the monounsaturated/polyunsaturated fatty acid profiles. The technological and biochemical potential of Bifidobacterium and Lactobacillus co-cultures, with or without inulin, on goats' and ewes' milk was assessed. Microbial stability, lactose consumption, organic acid production, proteolytic parameters and conjugated linoleic acid (CLA) production in situ, were followed in ewes' and goats’ fermented milk (EFM and GFM, respectively) over 21 days at 4 °C; technological feasibility for probiotic fermented milk production was shown. In EFM, all co-cultures presented high viable cell numbers (>7.0 log cfu mL⁻¹) throughout storage, presenting faster acidification capacities and higher CLA isomer levels than in GFM. Inulin had no impact on probiotic growth, yet contributed to storage stability. CLA isomers and proteolysis indices were co-culture dependent traits: for example, co-culture of Bifidobacterium animalis B94 with Lactobacillus acidophilus L10 registered the best CLA-production in GFM.