Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

Introduction: The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. Methods: We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Results and discussion: Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Biofilm formation by heat-resistant dairy bacteria: multispecies biofilm model under static and dynamic conditions.

In the food industry, especially dairy, biofilms can be formed by heat-resistant spoilage and pathogenic bacteria from the farm. Such biofilms may persist throughout the processing chain and contaminate milk and dairy products continuously, increasing equipment cleaning, maintenance costs, and product recalls. Most biofilms are multispecies, yet most studies focus on single-species models. A multispecies model of dairy biofilm was developed under static and dynamic conditions using heat-resistant Bacillus licheniformis, Pseudomonas aeruginosa, Clostridium tyrobutyricum, Enterococcus faecalis, Streptococcus thermophilus, and Rothia kristinae isolated from dairies. C. tyrobutiricum and R. kristinae were weak producers of biofilm, whereas the other four were moderate to strong producers. Based on cross-streaking on agar, P. aeruginosa was found to inhibit B. licheniformis and E. faecalis. In multispecies biofilm formed on stainless steel in a CDC reactor fed microfiltered milk, the strong biofilm producers were dominant while the weak producers were barely detectable. All biofilm matrices were dispersed easily by proteinase K treatment but were less sensitive to DNase or carbohydrases. Further studies are needed to deepen our understanding of multispecies biofilms and interactions within to develop improved preventive strategies to control the proliferation of spoilage and pathogenic bacteria in dairies and other food processing environments. IMPORTANCE A model of multispecies biofilm was created to study biofilm formation by heat-resistant bacteria in the dairy industry. The biofilm formation potential was evaluated under static conditions. A continuous flow version was then developed to study multispecies biofilm formed on stainless steel in microfiltered milk under dynamic conditions encountered in dairy processing equipment. The study of biofilm composition and bacterial interactions therein will lead to more effective means of suppressing bacterial growth on food processing equipment and contamination of products with spoilage and pathogenic bacteria, which represent considerable economic loss.

Graduate Student Literature Review: Farm management practices: Potential microbial sources that determine the microbiota of raw bovine milk.

Environmental and herd-associated factors such as geographical location, climatic conditions, forage types, bedding, soil, animal genetics, herd size, housing, lactation stage, and udder health are exploited by farmers to dictate specific management strategies that ensure dairy operation profitability and enhance the sustainability of milk production. Along with milking routines, milking systems, and storage conditions, these farming practices greatly influence the microbiota of raw milk, as evidenced by several recent studies. During the past few years, the increased interest in high-throughput sequencing technologies combined with culture-dependent methods to investigate dairy microbial ecology has improved our understanding of raw milk community dynamics throughout storage and processing. However, knowledge is still lacking on the niche-specific communities in the farm environment, and on the factors that determine bacteria transfer to the raw milk. This review summarizes findings from the past 2 decades regarding the effects of farm management practices on the diversity of bacterial species that determine the microbiological quality of raw cow milk.

Effect of two thermoresistant non-starter lactic acid bacteria strains on volatilome profile during Cheddar ripening simulation.

Dairy farm management practices can modify milk microbiota and therefore modulate non-starter lactic acid bacteria (NSLAB) found in cheese. These NSLAB can cause organoleptic defects. This study aimed to investigate the impact of two potential NSLAB in Cheddar cheesemaking: Lactiplantibacillus plantarum RKG 2-212 a strain isolated both in corn silage and raw milk, and Lactobacillus delbrueckii RKG R10, a strain isolated after pasteurisation of milk from a farm using grass and legume silage, and corn silage. The whole genome of these two lactobacilli was first sequenced. Then, the thermoresistance was evaluated after treatment at 60 °C for 5 min and compared to reference strains. Both lactobacilli were highly thermoresistant compared to other three lactic acid bacteria which are Lactococcus lactis subsp. cremoris ATCC 19257 and SK11, and L. plantarum ATCC 14917 (P < 0.0001). They lost less than 1 log cfu/mL (Δlog) and their genome contained a great number of copy number of genes coding for heat shock protein. During a Pearce test activity simulating Cheddar cheesemaking, the two lactobacilli did not show interaction with the starter Lcc. lactis subsp. cremoris SK11, and their population remained stable. During a ripening simulation, L. delbrueckii RKG R10 had a slight loss in viability in cheese slurry samples incubated at 30 °C for 12 d. However, L. plantarum RKG 2-212 had considerable growth, from 6.51 to 8.3 log cfu/g. This growth was associated with the acidification of the slurries (P < 0.0001). The presence of the lactobacilli modified the profile of volatile compounds evaluated by gas chromatography-mass spectrometry, accounting for 10.7% of the variation. The strain L. plantarum RKG 2-212 produced volatile compounds in greater quantity that could be associated with organoleptic defects such as acetic acid and 2-methylbutyraldehyde. Therefore, silage can be a vector of thermoresistant lactic acid bacteria for milk which can lead to flavor defects in cheese.

Optimization of bioprocess of Schleiferilactobacillus harbinensis Ca12 and its viability in frozen Brazilian berries (Açai, Euterpe oleracea Mart.).

Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional value and antioxidant activity and have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional potential, providing a source of carbohydrates, fibers, proteins, lipids, vitamins, and minerals. Furthermore, açai provides several health benefits, including antioxidant activity. Nutritionally enhanced foods have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Probiotics are dietary supplements consisting of live, beneficial microorganisms in the host which improve the intestinal microbiota. The objective of this study was to isolate, identify, and characterize the probiotic potential of an isolated Schleiferilactobacillus harbinensis strain (dubbed Ca12) and provide an optimized bioprocess for its production, using the complete factorial and central rotational compound design to supplement the frozen açai pulp. The isolated strain S. harbinensis Ca12 presented adequate resistance to gastric juice and bile salts, microbial activity against different Candida strains, self-aggregation and coaggregation properties, high adhesion in HT-29 cells, and 35% inhibition of Salmonella in HT-29 cells. When optimized, the cellular biomass production of the S. harbinensis Ca12 strain was approximately 600% higher than the unsupplemented whey, with a production of 3.6 × 1010 CFU mL-1. The S. harbinensis Ca12 strain's viability in the creamy and traditional frozen açai pulp was shown to be stable for up to 6 months at 20 °C. The impact of this study involved for the first time the S. harbinensis Ca12 described in the Brazilian cocoa pulp with activity against Candida albicans of clinical importance, creating the potential of a new functional food with important benefits to human health as prevention for candidiasis.

Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.

Lactic acid bacteria (LAB) found in milk can be responsible for organoleptic defects in cheese. To identify sources of LAB that could potentially develop during cheese making, we evaluated their prevalence and abundance in milk according to the type of forage used in dairy cow feeding. Forages and bulk tank milk were sampled 3 times on 24 farms using either hay alone (control), or grass or legume silage supplemented with corn silage or not. Both types of silage were either non-inoculated or inoculated with commercial preparations containing at least a Lactobacillus buchneri strain along with Lactobacillus casei, Lactobacillus plantarum, Enterococcus faecium, or Pediococcus pentosaceus. Our results indicate that LAB viable counts in milk samples (2.56 log cfu/mL) did not differ according to the type of forage used. A total of 1,239 LAB were isolated and identified by partial 16S rRNA gene sequencing. Although inoculation increased lactobacilli abundance in grass silage by 35%, we did not observe an effect on the LAB profile of milk. Indeed, we found no significant difference in milk LAB prevalence and abundance according to the type of forage (P > 0.05). Moreover, isolates belonging to the L. buchneri group were rarely found in bulk tank milk (3 out of 481 isolates). Random amplified polymorphic DNA typing of 406 LAB isolates revealed the plausible transfer of some strains from silage to milk (~6%). Thus, forage is only a minor contributor to LAB contamination of milk.

Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.

The dairy farm environment influences the raw milk microbiota and consequently affects milk processing. Therefore, it is crucial to investigate farm management practices such as the bedding materials. The aim of this study was to evaluate the effect of recycled manure solids (RMS) as bedding material on bulk tank milk and microbiological implications for cheese quality. Bulk tank samples were collected from 84 dairy farms using RMS or straw bedding. The use of RMS did not influence thermophilic and mesophilic aerobic viable counts from spores. However, straw-milk samples gave higher values for mesophilic anaerobic spore-forming bacteria (0.44 log cfu/mL) than RMS-milk samples (0.17 log cfu/mL). The presence of thermoresistant lactic acid bacteria was not increased in milk from farms using RMS. Nevertheless, taxonomic profiles of thermoresistant bacteria isolated were different between the 2 types of milk. More Enterococcus faecalis and Streptococcus spp. were identified in RMS-milk samples. Thermoresistant enterococci and streptococci could easily end up in cheese. Therefore, milk proteolytic activities of these isolates were tested. Neither Streptococcus spp. nor Enterococcus faecium isolates exhibited proteolytic activities, whereas 53% of E. faecalis showed some. Also, only 1 vancomycin-resistant enterococcus was detected. Survival of selected RMS-milk samples isolates (3 E. faecalis and 1 Streptococcus thermophilus) was evaluated during a model Cheddar cheese manufacture. Although those strains survived well, they did not modify the acidification curve of milk. However, they might cause organoleptic defects during cheese maturing.

Rapid screening of starter cultures for maari based on antifungal properties.

Forty Bacillus isolates obtained from maari (used as condiment in Burkina Faso) including 17 B. subtilis, 4 B. circulans, 7 B. pumilus and 6 B. licheniformis were investigated for use as starter cultures in maari production. The isolates were screened by PCR for the sfp gene responsible for the production of the lipopeptide biosurfactant, surfactin. The sfp gene was detected in all of the seventeen B. subtilis isolates, in 2 out of 7 B. pumilus, in 4 out of 6 B. licheniformis whereas no B. circulans was positive for the sfp gene by PCR screening. Furthermore, all the 40 Bacillus spp. were screened for biosurfactant production and inhibitory activity against Aspergillus flavus, A. niger, A. versicolor and Rhizopus oryzae. Results demonstrated a relationship between the presence of the sfp gene and the antifungal activity and biosurfactant production of Bacillus isolates. In addition, molecular typing of the 17 B. subtilis isolates by Multilocus Sequence Typing (MLST) resulted in 15 Sequence Types, one of them included three strains. Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), used for B. licheniformis, B. megaterium, B. circulans and B. pumilus revealed that the inhibitory activity and biosurfactant production were strain-dependent. Finally, the detection of chitinase (chi) and ß-glucanase (glu) biosynthesis genes was found to be associated with the antifungal activity for 16 B. subtilis isolates. The present work provides a greater understanding of the antifungal activity and biosurfactant production ability within the Bacillus spp. isolated from maari and contributes to the selection of Bacillus isolates to be used as starter cultures for controlled production of maari.

Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides.

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS.

Bioaccessible antioxidants in milk fermented by Bifidobacterium longum subsp. longum strains.

Bifidobacterium longum subsp. longum is among the dominant species of the human gastrointestinal microbiota and could thus have potential as probiotics. New targets such as antioxidant properties have interest for beneficial effects on health. The objective of this study was to evaluate the bioaccessibility of antioxidants in milk fermented by selected B. longum subsp. longum strains during in vitro dynamic digestion. The antioxidant capacity of cell extracts from 38 strains, of which 32 belong to B. longum subsp. longum, was evaluated with the ORAC (oxygen radical absorbance capacity) method. On the basis of screening and gene sequence typing by multilocus locus sequence analysis (MLSA), five strains were chosen for fermenting reconstituted skim milk. Antioxidant capacity varied among the strains tested (P = 0.0009). Two strains of B. longum subsp. longum (CUETM 172 and 171) showed significantly higher ORAC values than the other bifidobacteria strains. However, there does not appear to be a relationship between gene sequence types and antioxidant capacity. The milk fermented by each of the five strains selected (CUETM 268, 172, 245, 247, or PRO 16-10) did not have higher initial ORAC values compared to the nonfermented milk samples. However, higher bioaccessibility of antioxidants in fermented milk (175-358%) was observed during digestion.