Progress report for ONE21-390
The goal of this partnership project is to improve protocols ensuring the safety, consistency, and organoleptic properties of artisanal cheese produced by The Cellars at Jasper Hill. This project seeks to:
- Evaluate the impact of feeding practices (hay versus pasture) on microbial communities in raw milk and cheese, as determined by a combination of culture-independent and -dependent methods.
- Apply this information to investigate the potential relationship between microbial community composition and the safety and organoleptic properties of artisanal cheese.
- Foster knowledge exchange between partners, including: a) Advice on novel diagnostic methods for pathogens associated with raw milk; b) Training of personnel at the Jasper Hill microbiology laboratory on methods used in rapid bacterial diagnosis through quantitative polymerase chain reaction; c) Outreach to farms supplying raw milk to Jasper Hill; d) Internship opportunities and course development (Food Microbiology) for students in Sustainable Agriculture programs at the University of Rhode Island; e) Initial development of a comparative model (seafood – cheese) for the study of complex microbial communities in food.
The information developed in this research will aid Jasper Hill to develop a process for approving milk suppliers into their expanding artisanal cheese program.
Vermont has a well-deserved reputation for innovation in sustainable agriculture, wonderfully illustrated in an award-winning Artisanal Cheese industry (Paxson 2014). This industry, similar to other niche food industries (e.g. wine, oysters), relies on the unique characteristics provided by the environments in which the raw materials are produced, a quality referred to as “Taste of Place” or terroir (or merroir for oysters)(Teigen DeMaster et al. 2019). Much of the unique value of artisanal cheese is provided by the complex microbial communities (also called microbial ecosystems in this proposal) present in raw milk, and how the processes of rind washing and aging shape these cheese microbial ecosystems. On the other hand, microbial communities in raw milk could also include pathogens affecting food safety, including Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. The safety of artisanal cheeses to the human consumer is ensured by: 1) following farm practices that minimize or eliminate the presence of pathogen reservoirs in the farm system, and 2) screening the raw milk for pathogens of concern to humans. Therefore, the process of producing artisanal cheeses is both an art and a science, in which the raw ingredient (milk) is first screened for “microbial balance” (i.e. a diverse microbial community in which pathogens are minimized or absent), and then masterfully processed to produce safe and consistent cheeses with unique organoleptic profiles (Laithier & Percival, 2015).
There are many challenges to consistently achieving this delicate microbial balance in artisanal cheese. The microbial ecosystem in raw milk is influenced by numerous farming practices and environmental factors, including the feed, bedding, seasons, and climate, to name a few. There is, however, a limited understanding on how different farm practices contribute to the microbial communities in raw milk. By evaluating how specific farm practices at Jasper Hill and partner farms influence microbial communities in the raw milk used to make cheese, this proposed research addresses aspects of agriculture sustainability that are at the heart of the NE SARE mission: 1) “reduction of environmental and/or health risks in agriculture” through management of food safety in raw milk; and 2) “increasing farm productivity” by improving protocols at the farms that produce the milk and at the JH Microbiology Lab, ensuring the organoleptic quality and consistency of the cheeses produced by JH.
This proposed project is the product of conversations between Gomez-Chiarri and the JH team. Gomez-Chiarri, who, in addition to her passion for seafood, has a passion for artisanal cheese, was interested in deepening her knowledge in food microbiology and microbial ecology during a sabbatical leave. Inspired by a Jasper Hill event in which research on cheese microbiomes was featured, she approached Mateo Kehler to explore a potential collaboration. In this sabbatical, she hopes to expand her work studying microbial communities in marine organisms (Stevick et al. 2019; 2021) to the more controlled and repeatable microbial communities in artisanal cheese, while lending her expertise in genomics and development of diagnostic tools to the JH Microbiology Lab (Wilbur et al. 2012).
- - Producer
- - Producer
- - Producer
- - Technical Advisor (Educator and Researcher)
Objective 1: Evaluate the impact of feeding practices (hay versus pasture) on microbial communities in raw milk and cheese (planned May 2022 – May 2023).
Experimental approach. Jasper Hill recently purchased Andersonville Farm, with the goal of expanding their milk supply and determining if the production methods developed at JH with a small herd of 45 Ayrshires can be scaled up to a larger operation. Andersonville Farm houses 230 milking cows (Holsteins) that are fed on a diet consisting on dry hay from mid-September to mid-May. Part of this herd (about 2/3) was transitioned to pasture through Summer of 2021, returning to dry hay feeding in September. In mid-May 2022, one third of the Andersonville herd will continue on the current hay feeding regime, while the other two-thirds will be split in two groups, based on age and lactational status, and transitioned to pasture. Samples of raw milk from each of the three groups will be collected 4 times (0, 4, 8, and 12 weeks after transition, for a total of 24 samples - 3 groups x 2 replicate samples per group x 4 time points) for bacterial community composition analysis using amplicon sequencing of 16S rDNA gene libraries. Samples of milk and filters from the milk lines will also be tested following routine culture procedures used in the JH microbiology and associated laboratories, in order to detect and quantify the presence of targeted microbes known to affect milk and cheese safety and quality (e.g. E. coli, Staph, Listeria). Data collected for each milk sample at each time point will also include total amount of milk per herd, days in milk, somatic cell count, and other milk nutritional characteristics (protein, lactose, fatty acid profile, etc.).
In order to evaluate the effect of location on milk from cows on pasture, samples of raw milk will also be taken from the single bulk tank at the Michaud and Bakers farms, which house smaller herds of cows (mixed breeds, but mostly Holsteins), at two time points: the time of transition from barn to pasture (mid-May 2022) and another time point at the end of the summer (to coincide with last sampling at Andersonville). Depending on availability of funds and the cost of sequencing at the time, additional samples may be included from milk collected at other partner farms.
Milk collected at each time point from both the Andersonville and Michaud farms will be used to make separate batches of artisanal cheese. Cheese samples (n= 3 cheeses per group sampled in duplicate, 6 samples per milk replicate) will be taken 4 times during the aging process (96 samples; 6 cheese samples per replicate x 2 feeding regimes x 2 replicate milk samples per feeding regime x 4 time points) for amplicon analysis. Selection of the cheese type to focus on for these experiments (and therefore, aging duration) will be done by the expert cheesemakers at the Cellars based on the milk profile of the Andersonville and Michaud herds. Multivariate analysis will be used to determine the impact of feeding practice on: a) microbial community diversity; b) microbial community composition; and c) levels of particular isolates of safety concern or organoleptic potential as determined by culture. Factors to be included in the statistical models include sampling time and health parameters (e.g. somatic cell count).
Sample collection (May – September 2022 for milk, May 2022 – May 2023 for cheese): Herd feeding will be performed by farm personnel under the supervision of the Farm Manager (Hunnewell) using the procedures currently used in the Andersonville (confined group on hay) and other farms (pasture group). Confined cows are fed a Total Mixed Ration (TMR) that includes a blend of 1st and 2nd cut dry hay mixed with a mash of grain and minerals whetted with whey from JH's cheese production. Cows on pasture will be grazed on native mixed grasses and legumes, rotated on a 24-hour schedule. Milk will be collected twice per day in a Double 8 rapid exit Delaval milking parlor. The milking parlor and equipment will be cleaned after each of the two groups (dry hat TMR and pasture) is milked. Samples for microbiological analysis will be collected during the morning milking from the milk bulk tanks using sterilized bottles and transported to the microbiology lab at Jasper Hill for processing using routine procedures, with some minor modifications to allow for culture-independent analysis (Cosetta & Wolfe 2019; 2020). Metadata include average days in milk at each sampling time and somatic cell count, as well as data on herd health from routine monitoring.
Sample processing for amplicon analysis: Milk samples will be processed as previously described methods for milk microbiomes (e.g. Doyle et al. 2017 and more recent publications). DNA from each of the milk and cheese samples (min of 240, two technical replicates from each milk and cheese sample) will be performed using the Qiagen AllPrep PowerViral DNA/RNA Kit or similar. Following extraction, DNA samples will be transported from JH to URI and processed for 16S rDNA gene amplification (V3-V4 region), library preparation, and sequencing using procedures commonly used in the Gomez-Chiarri lab (Stevick et al., 2019; 2021). Positive and negative control samples will include a mock community sample and a blank control. Sequencing data will be processed and analyzed following the most updated procedures. Resulting data will include taxonomic assignments and Amplicon Sequence Variance counts, which will be used to determine bacterial diversity (Shannon, Simpson indices) and composition (taxonomic relative abundance).
Culture dependent methods: Samples of milk and cheese will be processed for culture in the JH microbiology laboratory using routine procedures (FDA’s Bacteriological Analytic Manual). These include plating of serial dilutions of samples on non-selective and selective media and bacterial identification using biochemical assays. Gomez-Chiarri will also train JH microbiologists in bacterial diagnosis using quantitative real time PCR (e.g. Cox & Gomez-Chiarri 2012; Gomez-Chiarri et al. 2015). The JH lab has recently acquired an Applied Biosystems qPCR instrument in order to have the capabilities for rapid microbial diagnosis (hours for qPCR versus one or more days for culture). Moreover, Petersson-Wolfe has arranged for loan of an instrument for the rapid diagnosis of pathogens associated with mastitis (Acumen Detection) and will advise on which other procedures may be available.
Objective 2: Investigate the potential relationship between microbial community composition and the safety and quality of artisanal cheese (January – August 2023).
Data collected in objective one will be analyzed and visualized to explore potential relationships between microbial community composition and the safety, organoleptic properties, and quality of artisanal cheese. As part of their normal operational procedures, JH will collect quality data on the cheeses made with the raw milk produced under the two different feeding practices (hay versus pasture) (Jasper Hill Cheese). Sequencing data will be transformed to account for the compositional data and analyzed using available statistical packages. The effect of different variables (feeding regime, sampling time, etc.) will be determined using non-parametric and multivariate statistics methods, such as NMDS analysis and general linear model (GLM) predictions in R. These methods will also allow to determine the effect of feeding regime on the presence and levels of selected microbial species, as determined by culture and/or qPCR. Other performance (i.e. cheese quality) parameters will be included in the multivariate statistics. A significant association of particular species with specific cheese quality parameters of interest will be confirmed using functional experiments in future research.
Overview of progress and description of changes to experimental approach (no changes to objectives):
In the original proposal, sampling of milk from the Andersonville Farm was scheduled for summer 2021. Based on timing of the funding, the experimental design was revised in Fall 2021 to delay the start of the sampling to May of 2022. In January 2022, Gomez-Chiarri (URI) and Madelynn Johnston (grazing manager for Jasper Hill Cellars) visited the two farms directly involved in the project, Andersonville and Michaud, to discuss the logistics of the sampling process with the farmers. Information from the visits was discussed with the Jasper Hill Cellars team and was used to revise the sampling design (described in more detail in revised methods above). The team also brainstormed the type of environmental, milk nutritional and microbial profile, and cheese properties data that will be collected at each sampling time point for each batch of milk and cheese. A preliminary set of samples will be collected in February - March 2022 to evaluate: a) the feasibility and ease of the milk sampling process; b) potential tank effects (the two bulk used at Andersonville differ in cooling structure); c) effect of two methods of teat cleaning; and d) optimize DNA extraction methods.
The logistics of splitting the herd at Andersonville Farm for the barn versus pasture were a little more complicated that originally expected, since cows are already split in three groups based on several factors (mainly age, lactational status). The experimental design was modified to try to account for these factors, as well as the limitation of just having two large milking tanks. The collection methods will be tested prior to the start of the pasture feeding in May.
Education & Outreach Activities and Participation Summary
Objective 3 will be focused on transfer of knowledge through a variety of outreach and extension efforts. Outreach will be facilitated by the URI/VT team of Gomez-Chiarri, Petersson-Wolfe, Deming, and Richard, whom have many years of experience in Outreach and Extension, and have received formal training in this area at Land Grant institutions.
Gomez-Chiarri and Consultants will directly transfer information and skills to the JH team through extended (several weeks at a time) visits by Gomez-Chiarri and interns and shorter visits by the Consultant Team (as needed). Jasper Hill has graciously offered rent-free housing for Gomez-Chiarri and the Consultant Team while performing research and outreach efforts. These efforts will include: (a) Providing advice to JH on novel diagnostic methods for pathogens associated with raw milk (Gomez-Chiarri and Petersson-Wolfe); (b) Training of personnel at the microbiology laboratory at JH in rapid bacterial diagnosis through quantitative polymerase chain reaction (Gomez-Chiarri).
Results from the research will be presented to farmers supplying raw milk to JH and other farmers potentially interested in transitioning their herds to pasture. This will be done through at least one Workshop to be held in Fall of 2022 (estimated attendance of about 10 – 20 farmers). This one-day workshop will include presentations from the JH and URI/VT Teams, as well as invited external experts (e.g. Food Safety regulators and extension agents). The workshop will cover subjects such as mastitis management (i.e. options for prevention and treatment or culling of the herd), cheese making procedures and the importance of raw milk quality in cheese quality. An emphasis will also be placed on avoiding the inappropriate use of antibiotics, a current interest of Gomez-Chiarri, who is a member of the National Academies of Sciences, Engineering, and Medicine Project on Examining the Long-Term Health and Economic Effects of Antimicrobial Resistance in the United States (2020 – 2022). The workshop will also include hands-on activities and tours of JH facilities. The workshop will be video recorded, and the materials will be assembled in a series of shorter (focused on key messages) videos to post at extension websites. The Consultant Team will also present the results from the research at regional and national conferences attended by dairy researchers, industry, and other stakeholders.
These efforts will aid in Jasper Hill fulfilling their desired long-term goal of gaining the ability to pool raw milk from various regions of Vermont to create pasture-based artisanal cheeses, resulting in the transition of more Vermont dairy operations to pasture-based production, and thus increasing the number of approved raw milk suppliers for artisanal cheese production.
Information and skill transfer will also occur from JH to URI, fostering the educational mission of this Land Grant institution. This partnership will lead to internship opportunities and course development (Food Microbiology – Gomez-Chiarri) for students in Sustainable Agriculture programs at URI. Finally, this partnership will aid in the initial development of a comparative model (seafood – cheese) for the study of complex microbial communities in food at the Gomez-Chiarri lab.