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)
- - Producer
Objective 1: Evaluate the impact of feeding practices (hay versus pasture) on microbial communities in raw milk and cheese (planned May 2022 – May 2023).
Jasper Hill recently purchased Andersonville Farm (Figs. 1 and 2), 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 about 230 milking cows (Holsteins) that were fed on a Total Mixed Ration (TMR) diet consisting on dry hay, a grain mix, and whey 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 March 2022, we did a preliminary study to: a) practice the logistics of milk sampling at Andersonville, b) collect samples to optimize DNA isolation from milk; and c) compare the effect of dry cleaning cow teats with wood wool, a more ecological method, as compared to iodine dips on microbial community (Figs. 3-5).
In early-May 2022, about half of the Andersonville herd continued on the current barn feeding regime, while the other half was transitioned to pasture. Herd feeding was performed by farm personnel under the supervision of the Farm Manager (Hunnewell) using the procedures currently used in the Andersonville farms. Confined cows were fed a Total Mixed Ration (TMR) including a blend of 1st and 2nd cut dry hay mixed with a mash of grain and minerals whetted with whey from Jasper Hill's cheese production. Cows on pasture were grazed on native mixed grasses and legumes, rotated on a 12-hour schedule. Pasture herds grazed for 12-hour intervals and were in the barn eating a TMR diet for the other 12 hours of the day.
Milk was collected twice per day in a Double 8 rapid exit Delaval milking parlor (Fig 3). The milking parlor and equipment were cleaned between each of the two groups (dry hay TMR and pasture) were milked. Samples for microbiological analysis were 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. Samples of raw milk from each of the two groups were collected 3 times (May 11, June 29, and September 9 2022, for a total of 12 samples - 2 groups x 2 replicate samples per group x 3 time points) for bacterial community composition analysis using amplicon sequencing of 16S rDNA gene libraries (URI) and culture-based methods (Jasper Hill). Samples of filters from the milk lines were also tested following routine culture procedures used in the Jasper Hill 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 spp.). Data collected for each milk sample at each time point also included total amount of milk per herd, days in milk, somatic cell count, and other milk nutritional characteristics (protein, lactose, fatty acid profile, etc.).
Milk collected at each time point (May, June, and September 2022) was used to make 3 separate batches of Alpha Tolman (Figs 6 and 7), an alpine type of artisanal cheese that ages for 8 – 9 months. The September 2022 cheeses will be ready around May - June 2023. Cheese samples (n= 3 cheeses per group – pasture or barn - collected in duplicate) are being taken 4 times during the aging process (total n = 144 samples) for sequencing analysis of the microbial community.
Samples of milk and cheese are being processed for culture in the Jasper Hill 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. The Jasper Hill microbiology laboratory is also implementing a qPCR assay for specific diagnosis of E. coli O157 in bacterial diagnosis using quantitative real time PCR (following FDA Bacterial Analytical Methods), based on preliminary data indicating the presence of this pathogen in some milk batches.
At URI, milk and cheese rind samples will be processed as previously described methods for milk microbiomes. DNA from each of the milk and cheese samples (two technical replicates from each milk and cheese sample) will be performed using the Qiagen PowerFood DNA Kit or similar. Following extraction, DNA samples will be processed for 16S rDNA gene amplification, library preparation, and sequencing using procedures commonly used in the Gomez-Chiarri lab. 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).
Objective 2: Investigate the potential relationship between microbial community composition and the safety and quality of artisanal cheese (March – 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, Jasper Hill will collect quality data on the cheeses made with the raw milk produced under the two different feeding practices (hay versus pasture). 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. Based also on the visits and limitations in the resources at each farm, the team decided to focus the study on just the Andersonville Farm.
The milk collection protocol was tested in March 2022. The team was interested in testing the use of wood wool, a dry and more environmentally sustainable method for cleaning cow teats before milking being used in the Swiss artisanal cheese. We determined, using a small number of cows (n = 4 per treatment, iodine or wood wool), that wood wool was less labor intensive than iodine dips, and that it had no significant impact on the microbiological food safety profile (as determined using culture dependent methods) of the milk. We are still processing the samples for 16S rRNA gene amplicon analysis, to be analyzed will all the other milk samples collected in May through September. Based on the results from the amplicon analysis, a decision will be made in further testing the use of wood wool.
The logistics for milk collection and making separate batches of cheese for pasture versus barn feeding were a little more complicated than originally expected. Therefore, the experimental design was modified to accommodate for the resources available for the project. The revision of the experimental plan is not expected to affect our ability to complete the objectives of the research.
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.