Bacterial Community Diversity and Structure Associated with the Cheese Rind and Curd of Seven Natural Rind Cheeses Lei Wei1‡, Rebecca J
Total Page：16
File Type：pdf, Size：1020Kb
CUTTING WEDGE: BACTERIAL COMMUNITY DIVERSITY AND STRUCTURE ASSOCIATED WITH THE CHEESE RIND AND CURD OF SEVEN NATURAL RIND CHEESES LEI WEI1‡, REBECCA J. RUBINSTEIN2‡, KATHLEEN M. HANLON2, HEIDI WADE1, CELESTE N. PETERSON3*, AND VANJA KLEPAC-CERAJ2* 1 DEPARTMENT OF CHEMISTRY, 2 DEPARTMENT OF BIOLOGICAL SCIENCES WELLESLEY COLLEGE, WELLESLEY, MA 2 DEPARTMENT OF BIOLOGY, SUFFOLK UNIVERSITY, BOSTON, MA ‡CO-FIRST AUTHORS: LEI WEI, REBECCA J. RUBINSTEIN MANUSCRIPT RECEIVED 30 JUNE 2016; ACCEPTED 5 NOVEMBER 2016 Copyright 2017, Fine Focus. All rights reserved. 10 • FINE FOCUS, VOL. 3 (1) ABSTRACT The microorganisms that inhabit cheese contribute greatly to the favor and development of the fnal product. While the rind and curd microbiota have been characterized separately, there is limited information on how the structure and function of microbial communities in rinds and curds vary within and amongst cheeses. To better understand the differences in community structure CORRESPONDING and function between communities of cheese rinds AUTHORS and curds, we combined culture-based methods with culture-independent community profling of curds and Vanja Klepac-Ceraj* rinds. Rinds contained greater taxonomic diversity than [email protected] curds. Lactobacillales dominated curd communities while members from the order Actinomycetales were found in high abundance in rind communities. Communities Celeste Peterson* varied more between rinds and curds than among [email protected] cheeses produced from different milk types. To better understand microbial community functions, we cultured and assayed isolates for antibiotic susceptibility and carbon source utilization. Among European and U.S. KEYWORDS cheeses, 70% of all susceptible isolates were cultured from U.S. cheeses. Overall, our study explored the • cheese microbiome differences within and between rind and curd microbial • rind communities of natural rind cheeses, provided insights • curd into the environmental factors that shape microbial • diversity communities, and demonstrated that at the community • antibiotic resistance and isolate level the cheese microbiome was diverse and • 16S rRNA gene metabolically complex. INTRODUCTION Cheese production exemplifes a humans optimized the cheese-making process reproducible succession of microbial to select for distinct and reproducible microbial communities (10, 48, 49). Microbes execute communities that give cheeses their individual the biochemical transformation of milk from tastes, consistencies, colors and other desirable a liquid suspension of lactose, casein, whey, properties (6, 14, 43, 49). In natural-rind and fat into cheese which is a solid aggregate cheeses, endogenous microorganisms or the of amino acids, lactate and volatile favor addition of bacterial starter culture to milk are compounds and pigments (6). Over centuries, needed to acidify and coagulate the milk into BACTERIAL COMMUNITY STRUCTURE IN CHEESE • 11 curds and enable future colonization by fungi lead to selection for more antibiotic-resistant and bacteria on the cheese surface (26, 49). bacterial strains (28). Likewise, bacteria on the The early process of curd formation is rind such as those belonging to the antibiotic- well characterized (11, 16) . Ripening begins producing Actinomycetes group can have a in milk, where lactic acid bacteria (LAB) such similar effect (32). The possibility of cheese as Streptococcus thermophilus, Lactobacillus bacteria developing resistance mechanisms has helveticus, and Lactobacillus casei ferment warranted the characterization of antibiotic- lactose into lactate, acidify the medium and susceptibility of bacteria on various types of digest proteins and milk (11, 25, 35, 36). LAB cheeses (4, 29). can be introduced by a starter culture, or the While many studies have characterized milk can be permitted to acidify naturally the succession of early cheese community from the microbial community in it (6). development from curd to rind in individual Regardless of the addition of starter culture, natural rind cheeses (1, 16, 20, 21), less is known most curds become inhabited by a simple about the comparison of microbial composition community, dominated by lactose fermenters of a mature rind to a mature curd within that may include organisms belonging to and across natural rind cheese varieties. Here genera Lactococcus, Lactobacillus, Leuconostoc, we compare microbial community structure Enterococcus, Staphylococcus, Enterobacter, and between the rinds and curds of seven natural- Streptococcus (1, 6, 16). rind cheeses that differ by pH, moisture After the initial curd formation, the rind content, and milk source. To obtain a detailed begins to form. Throughout the ripening, understanding of the rind and curd microbial excess lactate accumulates and dissolves into communities, we used culture-independent, the medium, eventually migrating upward high-throughput Illumina sequencing of 16S to the curd surface (22). Once lactate becomes rRNA genes. We also sought to characterize accessible at the outer surface of the cheese, specifc bacteria cultured from the cheeses. aerobic fungal taxa including Candida, These isolates were identifed using Sanger Penicillium, and Scopulariopsis colonize the sequencing of the 16S rRNA gene (40) and surface and metabolize lactate, leading to an were assayed for antibiotic susceptibility and increase in pH at the surface environment (22, carbon utilization profles using BIOLOG’s 49). De-acidifcation facilitates the colonization Ecoplates. Through these analyses, we aimed and succession of microbes that prefer to evaluate the following hypotheses: 1) cheese more alkaline and salty conditions including rind communities would exhibit higher coryneforms such as Corynebacterium, taxonomical diversity than curd communities; Brevibacterium and Brachybacterium (27, 37, 41). 2) antibiotic susceptibility of cheese isolates The composition of the external rind would differ between cheeses and between communities is governed by factors beyond the two regions where these cheeses originated, pH, including microbe-microbe interactions. Europe and the U.S.; and 3) cheeses with Fungal species can contribute to cheeses’ unique higher taxonomical diversity would be more characteristics, such as the blue-vein appearance metabolically active and can utilize more of Roquefort by the fungus Penicillium carbon sources. This study contributes to ro queforti, and can be crucial to the survival of the characterization of the curd- and rind- rind bacteria like Corynebacterium, Halomonas, associated communities of natural rind cheeses Pseudomonas, Pseudoalteromonas, and Vibrio and reveals patterns of microbial diversity spp. (4, 29, 49). Fungi such as those belonging according to cheese type as well as the overall to the spore-forming Penicillium species can metabolic and antibiotic resistance profle of also produce antibacterial compounds that can isolates from the different cheeses. 12 • FINE FOCUS, VOL. 3 (1) METHODS SAMPLE COLLECTION DNA ANALYSIS OF ISOLATES AND ENVIRONMENTAL The genomic DNA of selected bacterial PARAMETERS isolates were extracted by suspending a colony in polymerase chain reaction (PCR)-grade Seven natural rind cheeses—Vermont water and freezing for 20 min at -80°C and Shepherd, Stichelton, Sonnet, Missouri Truckle, then thawing. A 1465 base pair (bp) sequence Maggie’s Round, Comte, and Alpage Gruyere— of the 16S rRNA gene was PCR-amplifed were obtained from Wasik’s Cheese Shop in from genomic DNA (10-30 ng). Mastermix Wellesley, Massachusetts. A 100 +/- 10 mg reagents consisted of 1 μg/μL bovine serum sample from each of the rinds and curds of albumin, 200 μM dNTP mix, 1x buffer w/ the examined cheeses was collected aseptically MgCl2, 300 pM 27F primer (AGA GTT TGA and processed immediately. Rind samples were TCC TGG CTC AG), 300 pM 1492R primer scraped using a sterile razor blade and curd (ACG GCT ACC TTG TTA CGA CTT) (IDT samples were taken from the cheese center after Integrated DNA Technologies, Inc., Coralville, scraping off the exposed curd layer. A solid- IA) (30), and 0.2 U Takara Ex. Ta q polymerase (Takara Clontech, Mountain View, CA). DNA state pH meter (S175CD/BNC; Sensorex, Garden extracts from Escherichia coli and sterile PCR- Grove, CA) was used to determine the pH of grade water were used as positive and negative each cheese rind and curd. Samples were dried controls, respectively. The thermal cycler for 7 days and moisture was determined by program ran for 34 cycles in the following subtracting the dry weight of the sample from order: 1 cycle of initial denaturation (3 min at the original wet weight of a 1 g sample. 95°C); 32 cycles of denaturation (30 sec at 95°C), annealing (25-30 sec at 50°C), and extension BACTERIAL ISOLATION AND (1.5 min at 72°C); 1 cycle of fnal extension CHARACTERIZATION (10 min at 72°C); and hold (4°C). Amplicons were detected from banding patterns on 1.5% The 100 +/- 10 mg of fresh rind and curd agarose gel electrophoresis and quantifed on the samples were crushed using a pellet pestle and NanoDrop 2000 UV-Vis spectrophotometer diluted in sterile water to 10-3, 10-4, and 10-5 (NanoDrop Products, Thermo Fisher Scientifc, of their original concentrations. All diluted Inc., Wilmington, DE). Excess TA Q polymerase, samples were grown aerobically at room primer and nucleotides were precipitated temperature on three different media: Plate from the amplicon solution by adding Count Agar (PCA) with Milk (PCAM: 5g 1μL USB ExoSap-iT reagent (Affymetrix, Inc., Santa Clara, CA) to