Low-cost culture medium for biomass production of lactic acid bacteria with probiotic potential destined to broilers / Desarrollo de un medio de cultivo de bajo costo para la producción de biomasa potencialmente probiótica destinado a pollos parrilleros

Abstract The aim of this study was to evaluate different low-cost culture media for biomass production of 3 potential probiotic L. salivarius strains, which could be destined to broilers at farms. Different formulated media based on whey permeate (WP) supplemented with nitrogenous sources were evaluated: yeast extract (YE), whey hydrolysate (WH) and MnSO..H.O (Mn), MgSO..7H.O (Mg). The growth of each strain in the formulated media and the cost was compared with their growth and cost in commercial medium (MRS). L. salivarius DSPV008P did not grow adequately in any of the formulated media. On the other hand, addition of YE and Mn in the formulated media increased L. salivarius DSPV002P and L. salivarius DPSV011P growth. In contrast, WH and Mg addition increased the L. salivarius DSPV002P biomass only. L. salivarius DSPV011P was the only strain that had similar growth performance in MRS as in the selected medium: WP + YE 8g/l + Mn. In this sense, L. salivarius DSPV011P reached a biomass of 9.22 Log (CFU/ml) in the selected formulated medium, with a low-cost growth medium 12 times less than in MRS. Although the effect of supplements added to the culture medium on kinetic parameters are strain dependent, L. salivarius DSPV011P is the strain with the best technological characteristics, capable of growing in a medium based on a by-product of the dairy industry supplemented with YE and Mn and at a much less cost than in MRS medium.

Resumen El objetivo de este trabajo fue evaluar diferentes medios de cultivo de bajo costo para la producción de biomasa de 3 cepas potencialmente probióticas de L. salivarius, las cuales podrían ser destinadas a pollos parrilleros en las granjas. Para ello se evaluaron diferentes formulaciones basadas en permeado de suero de queso (WP) suplementado con fuentes nitrogenadas: extracto de levadura (YE) e hidrolizado de suero (WH) y MnSO..H.O (Mn), MgSO..7H.O (Mg). El crecimiento de las cepas en estas formulaciones y el costo económico fue comparado con el crecimiento y costo en el medio de cultivo comercial (MRS). L. salivarius DSPV008P no creció adecuadamente en ninguno de los medios evaluados. Por otro lado, la adición del YE y Mg al medio mejoró el desarrollo microbiano de L. salivarius DSPV002P y L. salivarius DPSV011P. El agregado de WH y Mn solo tuvo un efecto positivo en el incremento de la biomasa de L. salivarius DSPV002P. L. salivarius DSPV011P fue la única cepa que desarrolló la misma cantidad de biomasa en MRS y en el medio seleccionado WP + YE 8 g/L + Mn. L. salivarius DSPV011P logró un desarrollo de biomasa de 9.22 Log (UFC/ml) en el medio seleccionado con un costo económico 12 veces menor que en MRS. Aunque el efecto de los suplementos añadidos al medio de cultivo sobre los parámetros cinéticos depende de la cepa, L. salivarius DSPV011P es la cepa con mejores características tecnológicas, capaz de crecer en un medio a base de un subproducto de la industria láctea suplementado con YE y Mn y a un costo mucho menor que en MRS.

Risk Assessment of Lactobacillus salivarius W24 Used as "Other Substances"

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements sold in Norway. These risk assessments will provide NFSA with the scientific basis while regulating the addition of “other substances” to food supplements and other foods. "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional and/or physiological effect. It is added mainly to food supplements, but also to other foods. VKM has not in this series of risk assessments of "other substances" evaluated any claimed beneficial effects from these substances, only possible adverse effects. The present report is a risk assessment of Lactobacillus salivarius W24, and it is based on previous risk assessments and articles retrieved from a literature search. The risk of L. salivarius W24 was assessed for the general population. However, in previous assessments of “probiotics” published by VKM, concerns have been identified for specific groups. Therefore, the risk was assessed for the age group with immature gastro-intestinal microbiota (age group 0-36 months), population with mature gastro-intestinal microbiota (>3 years) and vulnerable groups independent of age. VKM has also assessed the risk of L. salivarius W24 in food supplements independent of the dose and have assessed exposure in general terms. Other sources of L. salivarius W24, such as foods, have not been included in the present risk assessment. VKM concludes that it is unlikely that L. salivarius W24 causes adverse health effects in the general healthy population with mature gastro-intestinal tract. However, no data on long-term adverse effects on infants and young children were identified. As evidence is accruing that the early microbial composition of the neonatal gut is important for the development of the gut microbiota and the immune system of the growing child, it is not possible to exclude that a daily supply of a single particular bacterial strain over a prolonged period of time to an immature gastro-intestinal tract may have long-term, although still unknown, adverse effects on that development.

Purification, characterization, mode of action, and enhanced production of Salivaricin mmaye1, a novel bacteriocin from Lactobacillus salivarius SPW1 of human gut origin

Background: Emergence of antibiotic resistance among pathogenic and food spoilage bacteria such as Staphylococcus aureus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mutans, Bacillus cereus, and Listeria monocytogenes triggered the search for alternative antimicrobials. An investigation aimed at purifying, characterizing, elucidating the mode of action, and enhancing the production of salivaricin from Lactobacillus salivarius of human gut origin was conducted. Results: Salivaricin mmaye1 is a novel bacteriocin purified from L. salivarius isolated from human feces. It is potent at micromolar concentrations and has a molecular weight of 1221.074 Da as determined by MALDI-TOF mass spectrometry. It has a broad spectrum of antibacterial activity. Salivaricin mmaye1 showed high thermal and chemical stability and moderate pH stability. The proteinaceous nature of salivaricin mmaye1 was revealed by the complete loss of activity after treatment with pepsin, trypsin, α-chymotrypsin, protease, and proteinase. Salivaricin mmaye1 is cell wall associated, and adsorption­desorption of the bacteriocin from the cell wall of the producer by pH modification proved successful. It exhibited a bactericidal mode of action mediated by pore formation. Its biosynthesis is regulated by a quorum sensing mechanism. Enhanced production of salivaricin mmaye1 was achieved in a newly developed growth medium. Conclusions: A novel, cell wall adhering, highly potent bacteriocin with a broad spectrum of inhibitory activity, membrane-permeabilizing ability, and enhanced production in a newly constituted medium has been isolated. It has a quorum sensing regulatory system and possesses interesting physicochemical characteristics favoring its future use in food biopreservation. These findings pave the way for future evaluation of its medical and food applications.

Effect of pH in the survival of Lactobacillus salivarius strain UCO_979C wild type and the pH acid acclimated variant

Background Bacterial acclimation involves cellular changes permitting the survival of a microorganism to prolonged acid pH exposure. The general aim of this work is to support this idea by determining the effect of pH in the survival of the human gastric derived probiotic strain Lactobacillus salivarius UCO_979C-1 (wild type) and L. salivarius UCO_979C-2 (acclimation to pH 2.6), which possesses anti-Helicobacter pylori properties. Results To assess this aim, the exopolysaccharide production through the phenol-sulfuric acid method was evaluated. Moreover, morphological and structural changes by transmission and scanning electron microscopy were observed. The bacterial survival was measured by viable count. The results showed that the acclimated variant strain synthesized higher levels of exopolysaccharide (690 ± 0.03 mg/L) more than the wild type (450 ± 0.12 mg/L). In addition, the acclimated variant preserved the viable count at pH 2.6 for 48 h, whereas the wild type strain decreases after 6 h and was non-viable at 24 h. Conclusion The results suggest that the acid stress acclimation of the strain L. salivarius UCO_979C-1 modified some cellular properties making this strain potentially useful as a gastric probiotic.

The influence of Lactobacillus salivarius on IgE,eosinophil cationic protein and transforming growth factor-βin serum and lung tissue homogenates of asthmic Balb/c mouse / 中国小儿急救医学

Objective To explore the influence of Lactobacillus salivarius on IgE,eosinophil cationic protein(ECP)and transforming growth factor(TGF)-βof serum and lung tissue homogenates of asthmic Balb/c mouse.Methods Thirty female Balb/c mice weighing 16-18 g(4 weeks old),SPF grade,were ran-domly divided into three groups:normal control group(group A),asthma group(group B),asthma combined Lactobacillus salivarius group(group C).Acute asthma model was established applying ovalbumin,behavioral changes were observed in mice,the changes in airway inflammation and the number changes of tracheal cilia were observed by pathological HE staining and the electron microscope respectively.The levels of IgE,ECP and TGF-βin the serum and lung tissue homogenate were measured by ELISA method.Results (1 )The mice in group B presented obvious restlessness,head and facial itching,scratching,shortness of breath,peri-oral cyanosis with abdominal breathing,sneezing,stooping,forelimbs shrinkage,urine and feces incontinence and other asthma acute phase performances in 5-1 0 minuts after 4% ovalbumin saline solution atomization excitation,the above reaction alleviated in group C compared to group B.(2)Pathology of HE staining in group B showed the thickening bronchial wall,narrow lumen,peribronchial and perivascular infiltration with a large number of eosinophil-based inflammatory cells,lumen with many inflammatory secretions,pathological changes in lung tissue of mice in group C significantly alleviated compared with group B.Under the electron microscope,the number of cilia in group B significantly decreased compared with group A,while group C significantly increased compared with group B.(3)Expression levels of serum IgE and ECP in group C were sig-nificantly lower than those in group B[(0.86 ±0.14)μg/ml vs.(1.30 ±0.12)μg/ml;(2.30 ±0.15)ng/ml vs.(3.41 ±0.15)ng/ml,P <0.05,respectively].The contents of lung tissue homogenate IgE[(0.97 ± 0.06)μg/ml]and ECP[(5.88 ±0.13)ng/ml]in group C significantly decreased,respectively,compared with the IgE[(1.13 ±0.05)μg/ml]and ECP[(8.08 ±0.15)ng/ml]in group B(P<0.05).The levels of serum TGF-β[(0.17 ±0.01 )μg/ml]and lung tissue homogenates TGF-β[(0.18 ±0.01 )μg/ml]in group C were higher than the serum TGF-β[(0.13 ±0.01 )μg/ml]and lung tissue homogenates TGF-β[(0.15 ±0.01 )μg/ml]in group B,respectively,the differences were statistically significant(P<0.05 ). Conclusion Lavage with Lactobacillus salivarius before sensitization could alleviate clinical symptoms of asthmatic mice,as well as reduce airway inflammation in some degree,TGF-βmay be involved in the mecha-nism of Lactobacillus salivarius playing a protective role.

Effect of Lactobacillussalivariuson the Number of CD4+CD25+Foxp3+Treg Cells and the Expression of TGF-β1 in Asthma Balb/c Mice / 中国医科大学学报

Objective To explore the effect of Lactobacillussalivariuson the number of CD4+CD25+Foxp3+Treg cells and expression of transform?ing growth factorβ1(TGF?β1)in asthma Balb/c mice. Methods Thirty?two female Balb/c mice were randomly divided into four groups:the nor?mal control group,the asthma group,the Lactobacillus salivarius group,and the asthma combined Lactobacillussalivariusgroup. Acute asthma mod?el was established by the ovalbumin challenge method. After extraction of primary spleen cells,flow cytometry was used to test CD4+CD25+Foxp3+Treg/CD4+T ratio in spleen lymphocytes. The levels of IL?4,IFN?γand TGF?β1 in the spleen cell culture supernatant were measured by ELISA method. Results The level of Th2 cytokine(IL?4)in the spleen cell culture supernatant of the asthma group was significantly higher than that of the control group(P<0.05),however,the level of Th1 cytokine(IFN?γ)was significantly lower than that of the control group(P<0.05). The ex?pression level of Th2 cytokine(IL?4)in the Lactobacillussalivariusintervention group was significantly decreased compared with the asthma group, and the Th1 cytokine(IFN?γ)expression level was elevated compared with the asthma group(P<0.05). The level of TGF?β1 in the Lactobacillus salivarius intervention group was higher than in the asthma group(P<0.05). The proportion of CD4+CD25+Foxp3+Treg/CD4+T in spleen lympho?cytes in the asthma group was lower than that in the control group(P<0.05),and was higher in the Lactobacillus salivarius intervention group than in the asthma group(P<0.05). Conclusion CD4+CD25+Foxp3+Treg was associated with the pathogenesis of asthma. Lactobacillus salivarius could adjust Th1/Th2 imbalance and reduce asthma inflammation through up?regulation of CD4+CD25+Foxp3+Treg and TGF?β1 expression.

Effect of lactobacillus salivarius on asthma Balb/c mouse in IL-4,IFN-γ and IgE of serum and lung tissue homogenates / 国际儿科学杂志

Objective To invstigate the effect of Lactobacillus salivarius on asthma Balb/c mouse in IgE,IL-4 and IFN-γ of serum and lung tissue homogenates.Methods Thirty female Balb/c mice weighing 16-18 g (4 weeks old), SPF grade, were randomly divided into three groups: normal control group (N), asthma group(A), asthma combined Lactobacillus salivarius group (AH).Acute asthma model was established by ovalbumin challenge method, mice were observed in behavioral changes, non-invasive method for the determination of airway responsiveness in mice, the changes in airway inflammation and the number changes of tracheal cilia were observed by pathological HE staining and the electron microscope.Respectively, the levels of IgE and IL-4/IFN-γin the serum and lung tissue homogenate were measured by ELISA.Results five ～ ten min after 4％ OVA saline solution atomization excitation mice in A group appeared obvious restlessness, head and facial itching, scratching, shortness of breath, perioral cyanosis with abdominal breathing, sneezing, stooping, forelimbs shrinkage, urine and feces incontinence and other asthma acute phase performances, The above reaction alleviated in AH group compared to A group;Pathology of HE staining in asthma group showed the thickening bronchial wall, narrow lumen, peribronchial and perivascular infiltration with a large number of eosinophil-based inflammatory cells,lumen with many inflannatory secretions,Pathological changes in lung tissue of mice in AH group significantly alleviated compared with asthma group;Under the electron microscope, the number of cilia in A group significantly decreased compared with N group, while AH group significantly increased compared with A group;Expression level of IgE,IL-4 in the serum and lung homogenate of A group was significantly higher than that of N group (P ＜ 0.05), the level of IFN-γexpression was significantly lower than N group (P ＜ 0.05), however the expression level of IgE, IL-4 in AH group significantly decreased compared with A group (P ＜ 0.05), and IFN-γexpression level elevated compared with A group(P ＜ 0.05).Conclusion Lavage with Lactobacillus salivarius before sensitization could alleviate clinical symptoms of asthmatic mice, partially correct Th1/Th2 imbalance as well as reduce airway infammafion.

In vitro antagonistic growth effects of Lactobacillus fermentum and Lactobacillus salivarius and their fermentative broth on periodontal pathogens

As lactobacilli possess an antagonistic growth property, these bacteria may be beneficial as bioprotective agents for infection control. However, whether the antagonistic growth effects are attributed to the lactobacilli themselves or their fermentative broth remains unclear. The antagonistic growth effects of Lactobacillus salivarius and Lactobacillus fermentum as well as their fermentative broth were thus tested using both disc agar diffusion test and broth dilution method, and their effects on periodontal pathogens, including Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalisin vitro at different concentrations and for different time periods were also compared. Both Lactobacillus salivarius and Lactobacillus fermentum and their concentrated fermentative broth were shown to inhibit significantly the growth of Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalis, althoughdifferent inhibitory effects were observed for different pathogens. The higher the counts of lactobacilli and the higher the folds of concentrated fermentative broth, the stronger the inhibitory effects are observed. The inhibitory effect is demonstrated to be dose-dependent. Moreover, for the lactobacilli themselves, Lactobacillus fermentum showed stronger inhibitory effects than Lactobacillus salivarius. However, the fermentative broth of Lactobacillus fermentum showed weaker inhibitory effects than that of Lactobacillus salivarius. These data suggested that lactobacilli and their fermentative broth exhibit antagonistic growth activity, and consumption of probiotics or their broth containing lactobacilli may benefit oral health.

Optimization of Growth medium for Efficient Cultivation of Lactobacillus salivarius i 24 using Response Surface Method

Production of Lactobacillus salivarius i 24, a probiotic strain for chicken, was studied in batch fermentation using 500 mL Erlenmeyer flask. Response surface method (RSM) was used to optimize the medium for efficient cultivation of the bacterium. The factors investigated were yeast extract, glucose and initial culture pH. A polynomial regression model with cubic and quartic terms was used for the analysis of the experimental data. Estimated optimal conditions of the factors for growth of L. salivarius i 24 were; 3.32 % (w/v) glucose, 4.31 % (w/v) yeast extract and initial culture pH of 6.10.