Improvement of shelf-life of cherry (Prunus avium L.) by combined application of modified-atmosphere packaging and antagonistic yeast for long-distance export.

BACKGROUND: The last decade has seen a growing interest in reducing the use of chemical fungicides for postharvest decay control. In the research for new, safe alternatives, the combined application of biocontrol agents and passive modified-atmosphere packaging (MAP) has been shown to be a promising strategy to extend fruit quality. Therefore, the aim of this work was to evaluate the effect of the combined application of MAP and two antagonistic yeasts, Metschnikowia pulcherrima L672 and Pichia kudriavzevii PK18, on sweet cherry shelf life. RESULTS: Microbiological, physico-chemical, and quality fruit analysis from batches treated with antagonistic yeast were compared with a control batch without yeast application and a batch to which fludioxonil (Scholar®) was applied. The composition of the atmosphere and physico-chemical traits showed similar values among the different batches during cold storage. However, interestingly, the combination of MAP with the antagonistic yeasts M. pulcherrima L672 and P. kudriavzevii PK18 increases the control of microbiological spoilage with results comparable to the application of fludioxonil. In addition, these batches experienced a slight decrease in volatile compounds associated with fresh fruit aroma, whereas in the control batch an increase of altered fruit aromas was observed. The same effect of control of spoilage was observed during the shelf life period. CONCLUSION: These results showed the positive effect of the combination of antagonistic yeasts and MAP, obtaining similar results in terms of control of microbiological spoilage and physico-chemical quality compared with the application of fludioxonil. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application.

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and ß-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.

Selection and application of antifungal VOCs-producing yeasts as biocontrol agents of grey mould in fruits.

Rotting caused by grey mould (Botrytis cinerea) is a concerning disease for numerous crops both pre- and postharvest stages. Application of antagonistic yeasts is a promising strategy for controlling grey mould incidence which could mitigate undesirable consequences of using synthetic fungicides. In this work, a screening for detection of yeasts isolated from figs producers of antifungal volatile organic compounds (VOCs) were performed by confrontation in double dishes systems. Eleven out of 34 yeasts confronted reduced B. cinerea growth parameter in vitro. This reduction was correlated (p ≤ 0.050) with the production of 10 volatile compounds: two acids (acetic acid and octanoic acid), 7 esters (Ethyl propionate, n-Propyl acetate, Isobutyl acetate, 2-methylbutyl acetate, furfuryl acetate, phenylmethyl acetate, 2-phenylethyl acetate) and one ketone (Heptan-2-one). In bases on in vitro assay, Hanseniaspora uvarum 793 was applied to in vivo assays with strawberries and cherries. The reduction of incidence of B. cinerea in strawberries at 7 °C and 25 °C was 54.9 and 72.1% after 6 and 3 days, respectively. The reduction of incidence of B. cinerea in cherries at 7 °C and 25 °C was 48.9 and 45.6% after 5 and 4 days, respectively. These results showed that VOCs produced by Hanseniaspora uvarum 793 are effective in the control of incidence of Botrytis cinerea in fruits, being a potential alternative to chemical fungicide.

Application of ultrasound for quality control of Torta del Casar cheese ripening.

This work aimed to establish the ultrasound parameters that can be useful to classify the defects in the soft cheese Torta del Casar during ripening. During ripening by ultrasound, 1 standard and 3 defective cheese batches (anomalous microbial population, inadequate pressing curd, and excessive pressing curd) were evaluated. Ultrasound parameters related to velocity, attenuation, and frequency were calculated and correlated with the physicochemical and rheological properties of the cheeses. Ultrasound data were considered variables in linear discriminant analysis to attempt cheese classification at different periods of the ripening process. Defective soft cheeses could be discriminated from standard ones with good accuracy, mainly at the final stages of ripening. The differentiation of cheese samples from 2 of the defective cheese batches (anomalous microbial population and inadequate pressing curd) from the standard was mainly attributed to different values of the attenuation-related parameters, whereas for samples from the other defective batch (excessive pressing curd), some parameters related to velocity and frequency were responsible for such discrimination.

Gene expression of Aspergillus flavus strains on a cheese model system to control aflatoxin production.

The expression of genes associated with aflatoxin biosynthesis by different Aspergillus flavus strains growing on a cheese model system has not been studied. To control aflatoxin biosynthesis, it would be useful to understand the changes in gene expression during cheesemaking and relate those changes to toxin production. The objective of this study was to evaluate the effects of pH, water activity, and temperature on the expression of 2 regulatory genes (aflR and aflS) and 1 structural gene (aflP) involved in aflatoxin biosynthesis, using 3 aflatoxigenic A. flavus strains growing on a cheese-based medium and reverse-transcription real-time PCR. The gene expression patterns were influenced by A. flavus strain and environmental conditions. The structural gene aflP and the regulatory genes aflR and aflS showed similar expression patterns in each A. flavus strain, but we also observed inter-strain differences. We observed the highest expression levels at 6 and 9 d of incubation by A. flavus strains CQ8 and CQ103, and saw a decrease in the days following. Strain CQ7 showed the lowest expression of these genes. We observed the highest expression levels of these genes at pH 5.5, water activity 0.95, and 20 to 25°C; strain CQ103 showed a different pattern for the aflS gene, with maximum expression at pH 6.0 on d 6 of incubation. For the 3 strains, we found a strong correlation between the relative expression of the aflR and aflS genes and the concentration of aflatoxins under conditions that simulated cheese ripening. Control strategies to avoid aflatoxin contamination during cheesemaking could use the detection of regulatory gene expression.

Characterization of microbial population of breba and main crops (Ficus carica) during cold storage: Influence of passive modified atmospheres (MAP) and antimicrobial extract application.

The purpose of this work was to study the changes of bacterial and fungal population of breba fruits such as 'Banane' and 'San Antonio' as well as 'Cuello Dama Negro', 'Cuello Dama Blanco' and 'San Antonio' fig cultivars stored in passive modified atmospheres (MAP) by the use of three different microperforated films (M10 with 16 holes; M30 with five holes and M50 with three holes). Moreover the effects of the application of aqueous soy polyphenolic antimicrobial extract (APE), alone or combined with MAP, were also studied for 'Cuello Dama Negro' and 'Cuello Dama Blanco' fig cultivars. Bacteria and fungi isolates were identified by PCR-RFLP of 16S rRNA and ITS regions, respectively, and subsequently sequence of the different patterns obtained. The results indicated that Pseudomonas gessardii, Pantoea agglomerans and Enterobacter asburiae were the main species of bacteria found in all the treatments studied. The fungal species identified were Aureobasidium pulullans, Cladosporium cladosporioides and Alternaria alternata, which were found in a lower percentage in fruit stored in MAP and fruits treated with antimicrobial extracts, as this treatments allowed to reduce the microbial growth of moulds and yeasts. Thus, the application of treatments such as M30, M50 or the combination of MAP with antimicrobial extract was highly effective to control fruit spoilage in fig and breba crops.

Bacterial communities of fresh goat meat packaged in modified atmosphere.

The objective of this work was to study the growth and development of fortuitous flora and food pathogens in fresh goat meat packaged under modified atmospheres containing two different concentrations of CO2. Meat samples were stored at 10 °C under two different modified-atmosphere packing (MAP) conditions: treatment A had 45% CO2 + 20% O2 + 35% N2 and treatment B had 20% CO2 + 55% O2 + 25% N2. During 14 days of storage, counts of each bacterial group and dominant species identification by 16S rRNA gene sequencing were performed to determine the microbial diversity present. The MAP condition used for treatment A was a more effective gas mixture for increasing the shelf life of fresh goat meat, significantly reducing the total number of viable bacteria and enterobacteria counts. Members of the Enterobacteriaceae family were the most common contaminants, although Hafnia alvei was dominant in treatment A and Serratia proteamaculans in treatment B. Identification studies at the species level showed that different microorganisms develop under different storage conditions, reflecting the importance of gas composition in the modified atmosphere on the bacterial community. This work provides new insights into the microbial changes of goat meat storage under different MAP conditions, which will be beneficial for the meat industry.

The growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors.

Traditional cheeses may be contaminated by aflatoxin-producing Aspergillus flavus during the ripening process, which has not been sufficiently taken into account. The objectives of this study were to evaluate the influence of water activity (aw), pH, and temperature on the lag phases, growth, and aflatoxin production of 3 A. flavus strains (CQ7, CQ8, and CG103) on a cheese-based medium. The results showed that the behavior of A. flavus strains was influenced by pH, aw, and temperature conditions. The CQ7 strain showed the maximum growth at pH 5.5, 0.99 aw, and 25°C, whereas for CQ8 and CQ103 strains, no differences were obtained at pH 5.5 and 6.0. In general, low pH, aw, and temperature values increased the latency times and decreased the growth rate and colony diameter, although aw and temperature were the most limiting factors. Maximum aflatoxin production on the cheese-based medium occurred at pH 5.0, 0.95 aw, and 25 or 30°C, depending on the strain. This study shows the effect of pH, aw, and temperature factors on growth and aflatoxin production of 3 aflatoxigenic A. flavus strains on a cheese-based medium. The findings may help to design control strategies during the cheesemaking process and storage, to prevent and avoid aflatoxin contamination by aflatoxigenic molds.

Antioxidant and antimicrobial activity of natural phenolic extract from defatted soybean flour by-product for stone fruit postharvest application.

BACKGROUND: Fresh fruit is highly perishable during storage and transport, so there has been growing interest in finding safe and natural antimicrobial compounds as a control tool. Phenolic compounds are secondary metabolites naturally present in vegetable material and have been associated with antimicrobial and antioxidant properties. Therefore, the aim of this study was to investigate the antioxidant capacity and potential antimicrobial effect of phenolic extract obtained from defatted soybean flour against selected pathogenic bacteria and microorganisms responsible of fruit decay. RESULTS: Analysis of phenolic composition by HPLC-MS showed the presence of a wide range of compounds, with isoflavones and phenolic acids the main polyphenols identified. Furthermore, the phenolic extract had important antioxidant activity by two different assays. Related to antimicrobial activity, in vitro experiments demonstrated that phenolic extract displayed a high activity against the main foodborne pathogens, while a moderate inhibition was found against five spoilage yeasts and Monilia laxa and a scarce effect for Penicillium glabrum, Cladosporium uredinicola and Botrytis cinerea. Interestingly these compounds considerably inhibited the mycelial growth of Monilia laxa, in both in vitro and in vivo experiments. CONCLUSION: The results of the present study revealed that defatted soybean flour is an important source of phenolic compounds with remarkable antimicrobial and antioxidant activity, suggesting the possibility of using them as natural additives in postharvest treatments to extend the shelf life of fruit.

Endo-ß-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins.

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-ß-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.

Impact of Simulated Human Gastrointestinal Digestion on the Functional Properties of Dietary Fibres Obtained from Broccoli Leaves, Grape Stems, Pomegranate and Tomato Peels.

This study aimed to analyse the impact of a simulated human digestion process on the composition and functional properties of dietary fibres derived from pomegranate-peel, tomato-peel, broccoli-stem and grape-stem by-products. For this purpose, a computer-controlled simulated digestion system consisting of three bioreactors (simulating the stomach, small intestine and colon) was utilised. Non-extractable phenols associated with dietary fibre and their influence on antioxidant capacity and antiproliferative activity were investigated throughout the simulated digestive phases. Additionally, the modifications in oligosaccharide composition, the microbiological population and short-chain fatty acids produced within the digestion media were examined. The type and composition of each dietary fibre significantly influenced its functional properties and behaviour during intestinal transit. Notably, the dietary fibre from the pomegranate peel retained its high phenol content throughout colon digestion, potentially enhancing intestinal health due to its strong antioxidant activity. Similarly, the dietary fibre from broccoli stems and pomegranate peel demonstrated anti-proliferative effects in both the small and the large intestines, prompting significant modifications in colonic microbiology. Moreover, these fibre types promoted the growth of bifidobacteria over lactic acid bacteria. Thus, these results suggest that the dietary fibre from pomegranate peel seems to be a promising functional food ingredient for improving human health.

Variation in consumption of human milk oligosaccharides by infant gut-associated strains of Bifidobacterium breve.

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates.

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding ß-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant ß-galactosidases from B. infantis ATCC 15697 displayed different preferences for ß-galactosides such as 4' and 6'-galactobiose, and four ß-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.

Role of the microbial population on the flavor of the soft-bodied cheese Torta del Casar.

The purpose of this work was to investigate the influence of the spontaneous microbial population on the flavor of Torta del Casar cheese. A total of 16 batches of cheeses with different microbial qualities were used. Their physicochemical and microbial characteristics were evaluated during ripening and then related with the volatile compounds, taste, and flavor properties of the finished cheeses. Acids were the most abundant volatile compounds, followed by alcohols and carbonyls. The amount of acetic acid and several alcohols were linked to cheeses with higher counts of lactic acid bacteria (LAB), whereas Enterobacteriaceae counts were associated with semivolatile fatty acids. The gram-positive catalase-positive cocci counts were correlated with esters and methyl ketones. Although the role of the LAB in the flavor development of Torta del Casar is the most relevant, other microbial groups are necessary to impart the flavor of the cheese and to minimize the possible off-flavor derived from excessive concentrations of LAB metabolites, such as acetic acid.

Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities.

Bifidobacteria are intestinal anaerobes often associated with gut health. Specific bifidobacterial species are particularly common in the gastrointestinal tract of breast-fed infants. Current short read next-generation sequencing approaches to profile fecal microbial ecologies do not discriminate bifidobacteria to the species level. Here we describe a low-cost terminal restriction fragment length polymorphism (TRFLP) procedure to distinguish between the common infant-associated bifidobacterial species. An empirical database of TRF sizes was created from both common reference strains and well-identified isolates from infant feces. Species-specific quantitative PCR validated bifidobacterial-specific TRFLP profiles from infant feces. These results indicate that bifidobacterial-specific TRFLP is a useful method to monitor intestinal bifidobacterial populations from infant fecal samples. When used alongside next generation sequencing methods that detect broader population levels at lower resolution, this high-throughput, low-cost tool can help clarify the role of bifidobacteria in health and disease.

Proteomic analyses of Bacteroides ovatus and Bifidobacterium longum in xylan bidirectional culture shows sugar cross-feeding interactions.

The intestinal microbiome is a community of anaerobic microorganisms whose activities significantly impact human health. Its composition can be modulated by consuming foods rich in dietary fiber, such as xylan, a complex polysaccharide that can be considered an emerging prebiotic. In this work, we evaluated how certain gut bacteria acted as primary degraders, fermenting dietary fibers, and releasing metabolites that other bacteria can further use. Different bacterial strains of Lactobacillus, Bifidobacterium, and Bacteroides were evaluated for their ability to consume xylan and interact with one another. Results from unidirectional assays gave indications of possible cross-feeding between bacteria using xylan as a carbon source. Bidirectional assays showed that Bifidobacterium longum PT4 increased its growth in the presence of Bacteroides ovatus HM222. Proteomic analyses indicated that B. ovatus HM222 synthesizes enzymes facilitating xylan degradation, such as ß-xylanase, arabinosidase, L-arabinose isomerase, and xylosidase. Interestingly, the relative abundance of these proteins remains largely unaffected in the presence of Bifidobacterium longum PT4. In the presence of B. ovatus, B. longum PT4 increased the production of enzymes such as α-L-arabinosidase, L-arabinose isomerase, xylulose kinase, xylose isomerase, and sugar transporters. These results show an example of positive interaction between bacteria mediated by xylan consumption. Bacteroides degraded this substrate to release xylooligosaccharides, or monosaccharides (xylose, arabinose), which might support the growth of secondary degraders such as B. longum.

Screening Microbial Interactions During Inulin Utilization Reveals Strong Competition and Proteomic Changes in Lacticaseibacillus paracasei M38.

Competition for resources is a common microbial interaction in the gut microbiome. Inulin is a well-studied prebiotic dietary fiber that profoundly shapes gut microbiome composition. Several community members and some probiotics, such as Lacticaseibacillus paracasei, deploy multiple molecular strategies to access fructans. In this work, we screened bacterial interactions during inulin utilization in representative gut microbes. Unidirectional and bidirectional assays were used to evaluate the effects of microbial interactions and global proteomic changes on inulin utilization. Unidirectional assays showed the total or partial consumption of inulin by many gut microbes. Partial consumption was associated with cross-feeding of fructose or short oligosaccharides. However, bidirectional assays showed strong competition from L. paracasei M38 against other gut microbes, reducing the growth and quantity of proteins found in the latter. L. paracasei dominated and outcompeted other inulin utilizers, such as Ligilactobacillus ruminis PT16, Bifidobacterium longum PT4, and Bacteroides fragilis HM714. The importance of strain-specific characteristics of L. paracasei, such as its high fitness for inulin consumption, allows it to be favored for bacterial competence. Proteomic studies indicated an increase in inulin-degrading enzymes in co-cultures, such as ß-fructosidase, 6-phosphofructokinase, the PTS D-fructose system, and ABC transporters. These results reveal that intestinal metabolic interactions are strain-dependent and might result in cross-feeding or competition depending on total or partial consumption of inulin. Partial degradation of inulin by certain bacteria favors coexistence. However, when L. paracasei M38 totally degrades the fiber, this does not happen. The synergy of this prebiotic with L. paracasei M38 could determine the predominance in the host as a potential probiotic.

Rapid discrimination of Bifidobacterium animalis subspecies by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Currently, the species Bifidobacterium animalis consists of two subspecies, B. animalis subsp. lactis and B. animalis subsp. animalis. Among these two subspecies, B. animalis subsp. lactis is especially important because it is widely used in the manufacture of probiotic dairy products. The application of these microbes in the food industry demands fast, accurate and low cost methods to differentiate between species and strains. Although various genotypic methods have been employed to discriminate between these two subspecies, they are not easily adapted for rapid identification in the industry. The purpose of this study was to evaluate the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to differentiate between the two subspecies of B. animalis, and for discrimination at strain level. We identified twenty-three strains of B. animalis at subspecies and strain level by genotypic methods and by proteomics using MALDI-TOF MS. The proteomics identification by MALDI-TOF was nearly identical to that obtained by genotypic identification using comparison of tuf and atpD gene sequences, and single-nucleotide polymorphisms (SNPs), insertions, and deletions (INDELs). We identified four protein markers, L1, L2, A1, and A2, which are useful for discriminating between both subspecies. Proteomics identification using MALDI-TOF MS was therefore an accurate method for discriminating and identifying these bacteria. Given the speed in which this method is achieved (~20 min including sample preparation), MALDI-TOF MS is promising as a tool for rapid discrimination of starter cultures and probiotics.

Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis.

Human milk contains high amounts of complex oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-D-glucosamine is a building block of these oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-ß-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human milk oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcß1-3 linkage found in lacto-N-tetraose, the most abundant human milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcß1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast milk. Bifidobacterium infantis N-acetyl-ß-D-hexosaminidases were induced during early growth in vitro on human milk oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human milk oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.

Characterization of autochthonal Hafnia spp. strains isolated from Spanish soft raw ewe's milk PDO cheeses to be used as adjunct culture.

The present work was performed to study the enterobacteria involved in the ripening of the artisanal raw ewe's milk PDO cheeses 'Torta del Casar' and 'Queso de la Serena' produced in Extremadura (Spain). These isolates were strain-typed, safety tested and characterized for some important technological properties. A total of 485 enterobacterial isolates were clustered by RAPD-PCR and subsequently identified by partial sequencing of the 16S rRNA gene. Among the 17 different species identified, Hafnia paralvei was the predominant species; H. alvei and Lelliottia amnigena were present to a lesser extent. Therefore, 55 Hafnia spp. strains, selected according to their genetic profile and dairy origin, were tested for the safe application. Overall, they were able to produce the biogenic amines putrescine and cadaverine under favourable conditions, presented α-haemolytic activity and did not produce cytolytic toxin active against HeLa cells or contain virulence genes. In addition, antibiotic susceptibility profiles showed that 17 Hafnia spp. strains were less resistant to the 33 antibiotics tested; subsequently, they were further technologically characterized. Although they showed differences, in general, they were well adapted to the stress conditions of cheese ripening. Among them, two strains, H. alvei 544 and 1142, are highlighted mainly due to their proteolytic activity at refrigeration temperatures and their low or null gas production. Although further studies are necessary before industrial application, these two strains are proposed for potential use as adjunct cultures to favour the homogeneity of these PDO cheeses, preserving their unique sensory characteristics.