Sensitivity of Molds From Spoiled Dairy Products Towards Bioprotective Lactic Acid Bacteria Cultures.

Fungal spoilage of dairy products is a major concern due to food waste and economical losses, some fungal metabolites may furthermore have adverse effects on human health. The use of lactic acid bacteria (LAB) is emerging as a potential clean label alternative to chemical preservatives. Here, our aim was to characterize the growth potential at three storage temperatures (5, 16, and 25°C) of a panel of molds (four Mucor and nine Penicillium strains) isolated from dairy products, then investigate the susceptibility of the molds toward 12 LAB cultures. Fungal cell growth and morphology in malt extract broth was monitored using oCelloScope at 25°C for 24 h. Mucor plumbeus 01180036 was the fastest growing and Penicillium roqueforti ISI4 (P. roqueforti ISI4) the slowest of the tested molds. On yogurt-agar plates, all molds grew at 5, 16, and 25°C in a temperature-dependent manner with Mucor strains growing faster than Penicillium strains regardless of temperature. The sensitivity toward 12 LAB cultures was tested using high-throughput overlay method and here all the molds except P. roqueforti ISI4 were strongly inhibited. The antifungal action of these LAB was confirmed when spotting mold spores on agar plates containing live cells of the LAB strains. However, if cells were removed from the fermentates, the inhibitory effects decreased markedly. The antifungal effects of volatiles tested in a plate-on-plate system without direct contact between mold and LAB culture media were modest. Some LAB binary combinations improved the antifungal activity against the growth of several molds beyond that of single cultures in yogurt serum. The role of competitive exclusion due to manganese depletion was examined as a possible antifungal mechanism for six Penicillium and two Mucor strains. It was shown that this mechanism was a major inhibition factor for the molds tested apart from the non-inhibited P. roqueforti ISI4 since addition of manganese with increasing concentrations of up to 0.1 mM resulted in partly or fully restored mold growth in yogurt. These findings help to understand the parameters influencing the mold spoilage of dairy products and the interactions between the contaminating strains, substrate, and bioprotective LAB cultures.

Shi C ，Knøchel S 《Frontiers in Microbiology》

Bioprotection Potential of Lacticaseibacillus rhamnosus LRH01 and Lactiplantibacillus plantarum LP01 against Spoilage-Associated Penicillium Strains in Yoghurt.

Shi C ，Knøchel S 《MOLECULES》

Lactic Acid Bacteria as Biopreservation Against Spoilage Molds in Dairy Products - A Review.

Mold spoilage of dairy products such as yogurt is a concern in dairy industry. Not only does it lead to substantial food waste, economic losses, and even brand image damage, but it may also cause public health concern due to the potential production of mycotoxin. Good hygiene practices are necessary to prevent contamination, but contamination may nevertheless occur at the production site and, not least, at the site of the consumer. In recent years, there has been a growing interest from consumers for "clean label" food products, which are natural, less-processed, and free of added, chemical preservatives, and a wish for shelf lives of considerable length in order to minimize food waste. This has sparked an interest in using lactic acid bacteria (LAB) or their metabolites as biopreservatives as a way to limit the growth of spoilage organisms in dairy products. A range of compounds produced by LAB with potential antifungal activity have been described as contributing factors to the inhibitory effect of LAB. More recently, growth inhibition effects caused by specific competitive exclusion have been elucidated. It has also become clear that the sensitivity toward both individual antifungal compounds and competition mechanisms differ among molds. In this review, the main spoilage molds encountered in dairy products are introduced, and an overview of the antifungal activity of LAB against different spoilage molds is presented including the main antifungal compounds derived from LAB cultures and the sensitivity of the spoilage molds observed toward these compounds. The recent findings of the role of competitive exclusion with emphasis on manganese depletion and the possible implications of this for biopreservation are described. Finally, some of the knowledge gaps, future challenges, and trends in the application of LAB biopreservation in dairy products are discussed.

Shi C ，Maktabdar M 《Frontiers in Microbiology》

Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Garnier L ，Mounier J ，Lê S ，Pawtowski A ，Pinon N ，Camier B ，Chatel M ，Garric G ，Thierry A ，Coton E ，Valence F ... -  《-》

Competitive Exclusion Is a Major Bioprotective Mechanism of Lactobacilli against Fungal Spoilage in Fermented Milk Products.

A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.

Siedler S ，Rau MH ，Bidstrup S ，Vento JM ，Aunsbjerg SD ，Bosma EF ，McNair LM ，Beisel CL ，Neves AR ... -  《-》