Development of probiotic beads similar to fish eggs

Probiotic foods are mainly restricted to dairy and soy products. This study aimed to develop a new probiotic beads similar to fish eggs, commonly used in oriental cuisine. Beads were produced by the extrusion encapsulation technique with calcium alginate, added to one of the following cultures: Lactobacillus rhamnosus GG ATCC 53103 and Bifidobacterium animalis DN-173 010 and stored for 30 days at 4 °C. The beads were characterized by the size, weight, morphology and viability of the probiotic strains in different storage temperatures and in simulated gastric juice adjusted to different pH values. The beads were also evaluated by a sensorial affective hedonic scale. The beads present a 2.8 mm diameter and a weight of 0.01 g (p > 0.05). Free and encapsulated cells were tolerant to pH 3.0. At pH 2.5 only of the encapsulated cells presented counts above 6 Log colony-forming units per gram (CFU/g). Beads containing L. rhamnosus showed higher viability 10⁷ CFU/g in storage for 30 days under refrigeration. The beads may be stored at abusive temperature for 5 h without loss of viability cells. The probiotic product developed showed an 82.2% acceptability index of overall characteristics and good market potential as a new probiotic product.     

Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage

The growth and metabolism of two probiotic organisms (L. acidophilus LAFTI^® L10 and Lactobacillus casei LAFTI^® L26) and a regular yoghurt culture (L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342) were studied in yoghurt containing 0.5%, 1.0%, and 1.5% (w/v) of high amylose corn starch powder (Hi-maize^®) or inulin. Viable cell counts of probiotic organisms, their metabolites and proteolytic activities, and viscosity of the yoghurts were determined during refrigerated storage for 28 d at 4 ^oC. In the presence of inulin, cultures showed better retention of viability (8.0 log cfu g⁻¹) in comparison with that of Hi-maize, which had a reduction by one log cycle. Lower concentrations of 0.5–1.0% Hi-maize improved (P<0.05) the production of propionic acid and also increased proteolytic activity of probiotic organisms substantially. A greater release of free amino acids may have sustained better growth of the organisms in yoghurts. Supplementation with either Hi-maize or inulin increased the viscosity of probiotic yoghurts significantly (P<0.05).     

Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation

The viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 microencapsulated by either an extrusion or an emulsion technique and used in white-brined cheese was monitored. Both microencapsulation techniques were effective in keeping the numbers of probiotic bacteria higher than the level of the therapeutic minimum (>10⁷ cfu g^?1). While the counts of probiotic bacteria decreased approximately 3 log in the control cheese in which probiotics were used as free cells, the decrease was more limited in the cheeses containing microencapsulated cells (approximately 1 log). Medium- and long-chain free fatty acid contents of the cheeses with immobilized probiotics were much higher than in the control cheese. Similarly, cheeses made with immobilized probiotics contained higher acetaldehyde and diacetyl levels than the control. Experimental cheeses containing microencapsulated probiotics were not different from the control cheese in terms of sensory properties.     

Flavour profiles and survival of starter cultures of yoghurt produced from high-pressure homogenized milk

Volatile carbonyl compounds, organic acids and yoghurt bacteria counts were investigated in yoghurts made from ultra-high pressure homogenized milk. Yoghurts were manufactured from milk treated using ultra-high pressure homogenization at 200 or 300 MPa and at 30 °C or 40 °C, and compared with those produced from heat-treated milk with 3% added skim milk powder. To study the evolution of these parameters, samples were analysed after days 1, 14 and 28 of storage. Yoghurts from milk heat-treated or treated at 300 MPa had very similar profiles of organic acids and volatile compounds, as well as similar bacterial counts of both starter cultures. In comparison, yoghurts from milk treated at 200 MPa at either 30 °C or 40 °C gave different profiles, together with a sharp decrease in counts of lactobacilli. During storage, only slight differences in flavour compounds and yoghurt bacteria counts were detected, except in those samples from milk treated at 200 MPa.     

Rapid enumeration of Bifidobacterium lactis in milk powders using impedance

An impedance method was evaluated to enumerate Bifidobacterium lactis, a probiotic, added to milk powder. Impedance changes were measured at 40 °C and recorded using the BacTrac™ 4100 microorganism growth analyser. Five different media were compared for the optimum impedance response. A raffinose-based medium (B. lactis medium) produced the fastest and most reproducible results. Good correlations were obtained between cell numbers from pure cultures of B. lactis (DR 10™) on reinforced clostridial agar plates and the impedance changes in the B. lactis medium. Enumeration of these bacteria in milk powder using the BacTrac™ 4100 impedance system showed no significant difference when compared with agar plate count results. The impedance counts estimated the cell counts of 10⁶ cells g⁻¹ within 15 h and was faster than the 3 days required to obtain a result using the agar plate count method.     

Assessment of antioxidant activity,lipid profile,general biochemical and immune system responses of Wistar rats fed with dairy dessert containing Lactobacillus acidophilus La-5

The viability and survival of Lactobacillus acidophilus La5 under in vitro simulated gastrointestinal in probiotic dairy dessert was assessed. In addition, the effects of regular consumption of the dessert (5 g/day) on the lipid profile, immune system, and antioxidant/biochemical status of Wistar rats were also evaluated after 2 weeks of treatment. Adequate counts of L. acidophilus La-5 were observed regards the viability and gastrointestinal conditions. The probiotic dairy dessert was efficient in reducing the LDL-cholesterol, triacylglycerol and increased the HDL-cholesterol in serum. Aspartate amino transferase, alanine aminotransferase, total protein, albumin, heat shock proteins, immune system responses, and blood-cells counts (monocyte, lymphocyte, neutrophil and leucocyte) were not affected (p > 0.05) after 15 days of treatment. Overall, the probiotic dairy dessert may be a viable alternative to enhance the blood lipid profile and could be used to improve the antioxidant defenses.     

Assessing the acid tolerance and the technological robustness of probiotic cultures for fortification in fruit juices

The suitability of probiotic cultures as fruit juice supplements was examined by assessing their acid tolerance and technological robustness. Survival of Lactobacillus and Bifidobacterium strains in orange juice (OJ), pineapple juice (PJ) and cranberry juice (CJ) was monitored. Results revealed that extensive differences exist among probiotic strains regarding their acid resistance. All of the strains screened survived for longer in OJ and PJ compared to CJ. L. casei DN-114 001, L. rhamnosus GG and L. paracasei NFBC43338 displayed the greatest robustness surviving at levels above 10⁷ cfu ml^− 1 in OJ and above 10⁶ cfu ml^− 1 in PJ for at least 12 weeks. Probiotic tolerance to thermal and non-thermal processing was studied to determine the feasibility of their addition to OJ prior to pasteurisation. OJ fortified with probiotic cultures was subjected thermal pasteurisation at 76 °C for 30 s and 90 °C for 1 min in addition to a high pressure treatment of 400 MPa for 5 min. Results indicated no strain was capable of withstanding treatments necessary to achieve a stable juice at levels > 10⁶ cfu ml^− 1. The outcome of the overall study points to L. rhamnosus GG, L. casei DN-114 001 and L. paracasei NFBC43338 as having promising potential for exploitation as functional supplements in fruit juices due to their impressive tolerance in acidic environments; however, fortification post processing is recommended.Industrial relevanceThe ability of health-promoting cultures to survive for at least 12 weeks in orange juice and pineapple juice at commercially critical levels renders them suitable strains for exploitation. Their inclusion may enhance the market potential of these already successful beverages.     

Temperature effect on the biological activity of Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251 in pure and mixed cultures grown in soymilk

The influence of temperature on the growth and biological activity of two probiotic strains (Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251) as pure and mixed cultures in soymilk (SM) were evaluated. Maximum growth was observed at 37°C in both mixed and pure cultures. In a product prepared with the mixed culture (1:1) at 37°C, the amount of lactic acid produced was approximately 55 mmol l⁻¹ after 24 h with a slow production rate (2.8 mmol l⁻¹ h⁻¹); the formation of acetic acid was higher with respect to pure cultures (82.01 mmol l⁻¹ after 24 h), and final pH (24 h) was 5.0. About 85% of the total amount of sugars in SM was reduced, mainly sucrose. Stachyose was reduced (71%) after 4 h of incubation. Maximum activity of alpha-galactosidase (alpha-gal) (13.2 U ml⁻¹) was observed after 6 h. At 37°C the bifidobacterium strain was viable in mixed culture throughout the period assayed. At lower (30°C) or higher (42°C) temperatures, mixed culture showed slower growth and lower acid production in SM but the alpha-gal activity was stimulated at 30°C.     

Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage

Experimentally prepared Jerusalem artichoke inulins (JAI) were compared with two commercial chicory root inulins for their prebiotic potentials in media broth model and growth-sustaining ability in non-fat yoghurts. Experimental yoghurts were made with 12% reconstituted skim milk (RSM) supplemented with 4% inulin powders, inoculated with mixed cultures of Lactobacillus casei LC-01, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (1:0.5:0.5 based on supplier’s recommendation) and incubated overnight at 37 °C. Non-supplemented yoghurt was prepared from 16% RSM and used as control. The survival and acidifying activity of lactic and probiotic cultures in all yoghurts were investigated on weekly intervals during the shelf life of 28 days at 4 °C. Incorporation of JAI resulted in improved viability of LC-01, maintaining >7.0 log CFU/g during cold storage but did not affect the viability of yoghurt bacteria in comparison with the control.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

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.     

Fermentation of beet juice by beneficial lactic acid bacteria

Red beets were evaluated as a potential substrate for the production of probiotic beet juice by four species of lactic acid bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus plantarum). All the lactic cultures were found capable of rapidly utilizing beet juice for cell synthesis and lactic acid production. However, L. acidophilus and L. plantarum produced a greater amount of lactic acid than other cultures and reduced the pH of fermented beet juice from an initial value of 6.3 to below 4.5 after 48 h of fermentation at 30°C. Although the lactic cultures in fermented beet juice gradually lost their viability during cold storage, the viable cell counts of these lactic acid bacteria except for L. acidophilus in the fermented beet juice still remained at 10⁶–10⁸ CFU/ml after 4 weeks of cold storage at 4°C.     

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.     

Effects of micronization on viability and thermotolerance of probiotic freeze-dried cultures

Three strains of probiotic freeze-dried bacteria, Bifidobacterium breve R070 (BB R070), Bifidobacterium longum R023 (BL R023), and Lactobacillus acidophilus R335 (LA R335), were micronized using a spiral jet mill as grinding system, in order to decrease the powder particle size for incorporation in multiphase low-diameter microcapsules produced by emulsification and spray-drying. The effects of grind air pressure (1, 2, 4 or 5.5 bar) and product feed rate (150 or 300 g h⁻¹) on powder particle-size distribution and bacterial viability were evaluated. The D(ν, 0.9) of the micronized powder was found to be only affected by grind air pressure (P<0.05). Survival of the micro-organisms to the particle-size reduction process was essentially related to the final powder particle size. A grind air pressure of 4 bar with a product feed rate of 300 g h⁻¹ was selected as the least destructive treatment (survival rate of 25.5±5.2%) to produce powders of probiotic freeze-dried cultures with particle-size distribution suitable for the microencapsulation technology (D(ν, 0.9)<25 μm). Heat susceptibility of the two micronized bifidobacteria strains prepared using these operating conditions was compared with that of the unprocessed cultures by means of various thermotolerance parameters (E_a, D_45°C, D_65°C, D_80°C). Results indicated that BL R023 displayed intrinsic low heat resistance whereas particle-size reduction dramatically increased the thermosensitivity of BB R070. Micronization is an effective way of reducing powder particle size of freeze-dried bacterial cultures for subsequent use in cell immobilization applications with low heat treatment.     

Effect of cryoprotectants,prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt

The survival of probiotic microorganisms including Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Bifidobacterium spp. was evaluated in yoghurt and freeze-dried yoghurt after processing and storage. The effectiveness of microencapsulating probiotic organisms as well as adding cryoprotectants and prebiotics in improving their viability was also investigated. The viability of Bifidobacterium infantis 17930 and L. rhamnosus GG was reduced by 0.07 log, while that of L. casei 1520 and Bifidobacterium longum 1941 was reduced by 0.28 and 0.39 log, respectively. There was a 7% improvement in the viability of L. casei 1520 when cryoprotectant ‘Unipectine™ RS 150’ was added at 2.5% (w/v). The prebiotic ‘Raftilose^®P95’ when added at 1.5% w/v to yoghurt improved the viability of the combined selected probiotic organisms by 1.42 log during four weeks of storage at 4 °C. Microencapsulation with alginate improved viability of combined selected probiotic organisms by 0.31 log in freeze-dried yoghurt stored at 21 °C.     

The protective effect of monosodium glutamate on survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 (E800) strains during spray-drying and storage in trehalose-containing powders

The use of trehalose as a means of preserving Lactobacillus rhamnosus GG (LGG) and L. rhamnosus E-97800 (E800) during spray-drying and the effects of incorporated monosodium glutamate (MSG) in the carrier medium on the survival rates during drying and storage were examined. E800 was more resistant to heat than LGG in 20%, w/w, trehalose; the d-values at 65 °C were 14 s and 5.1 s, respectively. An air outlet temperature of 65–70 °C was taken as optimal for the drying process, as the resultant moisture levels in trehalose containing these bacteria were 4.1% (w/w) and 3.79% (w/w) with corresponding viable counts of 3.65 × 10⁸ cfu mL^?1 and 1.80 × 10⁹ cfu mL^?1, respectively. The presence of MSG increased the final viable counts of LGG and E800 to 3.05 × 10⁹ cfu mL^?1 and 1.30 × 10⁹ cfu mL^?1, respectively. Survival of LGG and E800 remained constant at a minimum level of ～10⁸ cfu mL^?1 during storage at 25 °C in trehalose–MSG medium.     

Protective role of Lactobacillus fermentum R6 against Clostridium perfringens in vitro and in chicken breast meat under temperature abuse conditions

Six bacterial species were evaluated to determine their inhibitory effects on Clostridium perfringens in vitro (brain heart infusion broth) and in situ (chicken breast meat) under temperature abuse conditions (4 ± 1 °C for 12 h, followed by 7 h at 28 ± 1 °C and then 4 ± 1 °C for 53 h). During abusive storage, rapid growth of C. perfringens from vegetative cell and spore inocula was observed, exhibiting a 2.68–3.37 log CFU/mL (or g) increase in bacterial counts. In the presence of Pediococcus pentosaceus P1 or Lactobacillus fermentum R6, the counts of C. perfringens remained unchanged in the samples containing vegetative cells at the end of storage (P < 0.05); for those containing spores, the germination and outgrowth were also effectively inhibited, decreasing in bacterial counts of > 1.9 log CFU/mL (or g) compared to those of the control (P < 0.05). The pH of chicken meat was slightly declined by 0.09 in the presence of L. fermentum (P > 0.05), and the inhibitory effect against C. perfringens was ascribed to non-acid antimicrobial substances. These results indicate a potential solution for bio-protecting chicken meat from C. perfringens growth.Industrial relevanceClostridium perfringens is a common pathogen that contaminates meat and meat products, but the organism cannot multiply under cold chain conditions at 4 °C. However, it was reported that temperature abuses commonly occurred during the transportation, storage or retail display of the food chill chain. During the abusive storage, C. perfringens could grow rapidly, which may lead to food poisoning. It is a serious problem for food safety.In this study, Lactobacillus fermentum R6 was found to show effective inhibition on both the growth of C. perfringens vegetative cells and the germination and outgrowth of its spores in chicken meat (P < 0.05) under temperature abuse conditions, and also it had a minimal effect on the pH of the meat (P > 0.05). The results reveal a potential technology for bio-protecting chicken meat from C. perfringens growth.     

Fermentative ability of alginate-prebiotic encapsulated Lactobacillus acidophilus and survival under simulated gastrointestinal conditions

Lactobacillus acidophilus was encapsulated in alginate-inulin-xanthan gum and its ability to grow in carrot juice and survive 8 weeks of storage at 4 °C and subsequent exposure to artificial gastrointestinal conditions were assessed. Encapsulation significantly enhanced cell viability after fermentation and storage (6 × 10¹² and 4 × 10¹⁰ cells/ml versus 4 × 10¹⁰ and 2 × 10⁸ for free cells, respectively). Encapsulation protected L. acidophilus from exposure to simulated gastric conditions; minor alterations in viability and the protein profile occurred after incubation in pancreatic juice. For free cells, viability decreased significantly and the expression of numerous proteins was lost after incubation in gastric and pancreatic juice. Thus, encapsulation preserved probiotic bacterial viability and activity; the addition of inulin as a prebiotic component could enhance the functional properties of food products containing this formulation.     

Safety aspects of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium animalis subsp. lactis HN019 in human infants aged 0–2 years

Given the relatively immature state of the neonatal gut and gut-associated immune system, the safety of probiotic strains for use as ingredients in infant milk formulae must be demonstrated in infant populations. As part of a double-blind placebo-controlled clinical trial of two commercially available probiotic strains in the reduction of risk for infant eczema, a number of safety outcomes were measured. Infants received daily doses of Lactobacillus rhamnosus HN001 (6 × 10⁹ cfu day^?1) or Bifidobacterium animalis subsp. lactis HN019 (9 × 10⁹ cfu day^?1), or placebo from birth to 24 months. Mothers received the same treatment from 35 weeks gestation, for up to 6 months postnatally while breastfeeding. No statistically significant differences were observed between the treatment groups for study withdrawal, incidence of adverse events, morphometric data, wheeze, and antibiotic use over the treatment period. We conclude that probiotics strains HN001 and HN019 were safe and well tolerated in infants, and did not affect normal growth.     

Effects of probiotic administration in swine

In the present work we evaluated the effects of probiotic strains administration in pigs. On the 35th day of age, 30 pigs were distributed into 2 groups: the non-treated control group (initial average BW: 8.3 ± 0.6 kg) and a probiotic supplemented fed group (initial average BW: 8.7 ± 0.4 kg). Each experimental group was fed ad libitum on a commercial diet with free access to tap water for 35 days. A mixed probiotic culture (10⁸ CFU/ml) was orally delivered, every day, to the animals of the probiotic supplemented fed group. Body weight (BW), feed intake (FI), efficiency (BW: Feed), and faecal microflora, were studied before and throughout the experimental trial. At the end of the fifth week, 5 animals of each dietary treatment were slaughtered and intestinal samples were taken for histology. The results obtained showed that the group receiving probiotic bacteria exhibited lower FI values and better efficiency than control group (P ≤ 0.05), but mean final BW values were not significantly different. Only significant changes (P ≤ 0.05) were found in enterobacteria population between control and probiotic supplemented fed group during the experimental period. By histological techniques it was observed that the treatment group has intestinal morphological structures more preserved than control group. These results suggest that probiotic bacteria administrated in this study could be used widespread as a way to improve growth performance parameters of animals avoiding the use of antibiotics as growth-promoting factors.