Article pubs.acs.org/JAFC
An Overview on the Presence of Cyclopropane Fatty Acids in Milk and Dairy Products Augusta Caligiani,* Angela Marseglia, and Gerardo Palla Dipartimento di Scienze degli alimenti, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy ABSTRACT: A survey was carried out to determine the presence of cyclopropane fatty acids (CPFA) in various dairy products. CPFA such as lactobacillic acid and dihydrosterculic acid are components of bacterial membranes and have been recently detected in milk from cows fed with maize silage. In this paper about 200 dairy samples comprising cow, sheep, and goat milk, cheese, yogurt/fermented milk, and butter were analyzed. Results showed that cow milks were generally positive to CPFA (0.014−0.105% of total fatty acids), while goat, yak, and sheep milks were negative. Experimental yogurt and fermented milks showed the same CPFA content of the starting milk. Positive to CPFA were also the majority of samples of commercial butter and cheeses, except some PDO cheeses as Parmigiano-Reggiano and Fontina, cheeses from mountain regions, and goat and sheep cheeses. These data suggest that the presence of CPFA in dairy products could be used as a marker of silage feeding. KEYWORDS: cyclopropane fatty acid, milk, butter, cheese, yogurt, fermented milk
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INTRODUCTION Although fatty acids containing three-carbon cyclic rings occur infrequently in plants, they are present in Malvaceae, Sterculiaceae, and Sapindaceae, representing a significant component of Litchi chinensis seed oils.1,2 Cyclopropane fatty acids (CPFA, Figure 1) are known to be components of
We recently reported the presence of CPFA in milk and cheeses, with amounts reaching 0.12% of the total fatty acids content:9 in this study we showed that milk samples from farms using animal feeding diets without maize silage do not contain cyclopropane fatty acid, while milk samples from farms that used maize silage were always positive for cyclopropane fatty acids. This trend was also observed in the corresponding cheeses. Moreover, feeds were also tested for CPFA presence, showing that maize silage samples were all positive for the cyclopropane fatty acid, while the other feed components as hay and grains were negative.9 Therefore, we considered that the presence of CPFA in milk is related to their presence in ensiled products. Crop ensiling technology is based on the natural fermentation of plant tissue juice mediated by the lactic acid bacteria (LAB) naturally present in the plant leaves. LAB convert soluble carbohydrates to organic acids, mainly lactic acid, under anaerobic conditions, and as a consequence the pH drops from 6.0−6.5 to 5.0−3.7.10 The microbial ecosystem of silage is dominated by LAB with the homofermentative Lactobacillus plantarum being the most frequently isolated from silage in general, but heterofermentative species (i.e., Lactobacillus buchneri and Lactobacillus brevis) are common.11−13 Brusetti et al. (2006) showed that Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after 6 days of fermentation. Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population.14 CPFA presence in silages can be therefore attributed to the microbial fermentations. However, many aspects regarding the incorporation of CPFA in milk triglycerides have to be clarified and additional information is needed on the presence and diffusion of CPFA in milk and dairy products. Therefore, the aim of this paper is to provide a
Figure 1. Structures of the main cyclopropane fatty acids (CPFA).
bacterial membranes. cis-11,12-Methyleneoctadecanoic acid was first isolated from the phospholipids of Lactobacillus arabinosus and given the trivial name of lactobacillic acid. It has also been found in a wide range of bacterial species, both Gram-negative and Gram-positive, and it is often accompanied by cis-9,10methylenehexadecanoic acid and other homologues. Some organisms contain cis-9,10-methyleneoctadecanoic acid (dihydrosterculic acid), derived from oleic acid, together with homologous fatty acids (C16 or C20 in chain length). The first step in the biosynthesis of the cyclopropane ring involves the addition of a methyl group to the precursor fatty acid double bond by S-adenosylmethionine.3 The bacterial production of cyclopropane ring is related to changes in the membrane fatty acid composition and represents one of the most important adaptive microbial response to stress exposure, together with the synthesis of specific proteins.4,5 In fact, CPFA are reported to favor the stress tolerance of several bacteria such as Lactobacillus helveticus, Lactobacillus bulgaricus, and Lactobacillus acidophilus.6 They are also cellular components of Lactobacillus sanf ranciscensis, which habitat is represented by cereal sour dough.7,8 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 7828
January 9, 2014 July 17, 2014 July 17, 2014 July 17, 2014 dx.doi.org/10.1021/jf4057204 | J. Agric. Food Chem. 2014, 62, 7828−7832
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Table 1. Presence of CPFA Detected by GC-MS Analysis in Milk Samplesa percentage of CPFA in positive samples raw bulk milk of different origin Lombardy (Italy) Veneto (Italy) Emilia Romagna (Italy) Piedmont (Italy) Austria Germany commercial cow milk samples (Parma market, Italy) goat milk (Parma market, Italy) sheep milk (Parma farms, Italy) yak milk (China)
no. of samples
no. of samples positive to CPFAb
mean ± SDc
min − maxc
6 5 2 2 2 5 6 6 6 10
6 5 2 2 1 2 4 0 0 0
0.080 ± 0.009 0.028 ± 0.009 0.038 ± 0.006 0.067 ± 0.002 0.065 ± 0.001d 0.039 ± 0.002 0.06 ± 0.03 nd nd nd
0.065−0.093 0.014−0.039 0.030−0.045 0.062−0.070 0.030−0.045 0.031−0.105
Results are reported as mean ± SD, minimum and maximum for homogeneous groups of samples. bPositivity for values ≥0.01% of the total fat content. cWith respect to the total chromatographic area of fatty acid methyl esters. dMean ± SD of replicate analyses of the only positive sample. a
survey on the presence of CPFA in dairy samples from different animal sources such as milk, cheeses, yogurt, other fermented milks, and butter, in order to obtain a deeper knowledge on the mechanisms involved in the transfer of CPFA from the bacterial cell walls to milk fat.
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Table 2. Presence of CPFA Detected by GC-MS Analysis in Fermented Milk Samplesa percentage of CPFAb winter starting milk
summer starting milk
Pasteurized Milk 0.038 ± 0.005 0.043 ± 0.003 Milk Fermented with Bifidobacterium infantis + Lactobacillus acidophilus zero time 0.044 ± 0.004 0.044 ± 0.005 10 days 0.042 ± 0.003 0.036 ± 0.004 20 days 0.041 ± 0.005 0.047 ± 0.001 30 days 0.037 ± 0.003 0.052 ± 0.006 Milk Fermented with Lactobacillus paracasei zero time 0.039 ± 0.002 0.042 ± 0.004 10 days 0.037 ± 0.002 0.039 ± 0.003 20 days 0.040 ± 0.003 0.044 ± 0.005 30 days 0.038 ± 0.003 0.047 ± 0.004 Milk Fermented with Lactobacillus bulgaricus + Streptococcus thermophilus (Yogurt) zero time 0.038 ± 0.002 0.042 ± 0.005 10 days 0.036 ± 0.004 0.041 ± 0.004 20 days 0.043 ± 0.004 0.047 ± 0.003 30 days 0.030 ± 0.003 0.039 ± 0.004 Milk Fermented with Lactococci zero time 0.040 ± 0.004 0.041 ± 0.004 10 days 0.035 ± 0.003 0.038 ± 0.003 20 days 0.036 ± 0.004 0.051 ± 0.006 30 days 0.034 ± 0.003 0.045 ± 0.004
MATERIALS AND METHODS
Sampling. Milk Samples (Table 1). Samples of bovine bulk milk were collected from stables in different Italian regions (Lombardy, Veneto, Emilia Romagna, Piedmont), Austria, and Germany and provided by Parmalat Spa. Six different brands of commercial milks, two of them coming from Italian alpine regions, were purchased on the market, together with six samples of goat milk. Six samples of sheep milk were from local farms (Parma). Additionally, ten samples of yak (Bos grunniens) milk coming from China were provided by Animal Science Department of Qinghai University, China. Yogurt and Fermented Milk (Table 2). Parmalat Spa (Parma, Italy) provided the experimental milks fermented with the following combination of microorganisms. Fermentations were conducted at 42 °C for 12 h. Trial A: Bif idobacterium infantis + Lactobacillus acidophilus Trial B: Lactobacillus paracasei Trial C: Lactobacillus bulgaricus + Streptococcus thermophilus (Yogurt) Trial D: Lactococci Each milk sample was analyzed before the addition of bacteria, after the inoculum, and then sampled at 10, 20, 30 days of the shelf life (stored refrigerated at 4 °C). Two series of experiments were conducted, utilizing bulk milk samples collected in winter or in summer. Cheese Samples. A total of 125 cheese samples were analyzed for their CPFA content. Samples of grated Parmigiano-Reggiano (10 samples) and Grana Padano cheeses (10 samples) were purchased on the market. Additionally, 15 samples of certified Grana Padano and 15 samples of certified Parmigiano-Reggiano were provided by Consorzio del Formaggio Parmigiano-Reggiano (Reggio Emilia, Italy). Ten samples of Asiago cheese from lowland and from highland farms were provided by Padua University (Italy). Other cheese samples coming from milk of different species (cow, goat, and sheep) having different production technologies (Table 3) were purchased on the market. Butter. Seven samples of commercial butter were purchased on the market, including one sample from a farm producing milk for Parmigiano-Reggiano cheese, one from an alpine farm, and one from goat milk. Sample Preparation for GC-MS Analysis of Milk Fat and Cheese. Ten grams of milk/fermented milk was mixed for 1 min with 1 mL of 33% ammonia (Carlo Erba, Milan, Italy). The mixture was then extracted twice with 20 mL of hexane/acetone (2/1 v/v) (SigmaAldrich, Saint Louis, MO, USA) in a separatory funnel; the organic
Results are reported as mean ± SD of replicate analysis. bWith respect to the total chromatographic area of fatty acid methyl esters. a
phase was then recovered and evaporated to dryness under vacuum. As regards the cheese analysis, 0.5 g of grated sample was stirred with a hexane/acetone mixture (20 mL, 4/1 v/v) and then filtered on paper. The organic phase was evaporated to dryness. Butter was directly submitted to the following steps. 100 mg of fat recovered was dissolved in hexane (5 mL) and mixed for 1 min with 2 mL of KOH 5% (Carlo Erba, Milan, Italy) in methanol (Sigma-Aldrich, Saint Louis, MO, USA). After phase separation, the superior organic phase was injected (1 μL, split mode) on an Agilent Technologies 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent Technologies 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). A low-polarity capillary column (SLB-5 ms, Supelco, Bellefonte, PA, USA) was used. The chromatogram was recorded in the scan mode (40−500 m/z) with a programmed temperature from 40 to 280 °C. Peak identity of CPFA was confirmed by comparison 7829
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Table 3. Presence of CPFA Detected by GC-MS Analysis in Cheese and Butter Samplesa percentage of CPFA in positive samples cheese
no. of samples
no. of samples positive to CPFAb
mean ± SDc
15 15 10 10 2 3 2 4 6 3 10 3 3 4 2 4 2 3 2
0 15 0 10 2 3 0 4 0 3 5 3 3 0 0 0 2 3 2
nd 0.15 ± 0.04 nd 0.08 ± 0.01 0.06 ± 0.02 0.15 ± 0.09 nd 0.07 ± 0.04 nd 0.08 ± 0.03 0.04 ± 0.02 0.08 ± 0.05 0.09 ± 0.04 nd nd nd 0.039 ± 0.001 0.036 ± 0.009 0.029 ± 0.001
13 2 2
0 0 0
nd nd nd
3 2 7
0 0 4
nd nd 0.04 ± 0.02
cow cheeses Parmigiano-Reggiano (Emilia Romagna, Italy) Grana Padano (Lombardy, Italy) commercial grated Parmigiano-Reggiano (Emilia Romagna, Italy) commercial grated Grana Padano (Lombardy, Italy) Taleggio (Lombardy, Italy) Gorgonzola (Lombardy, Italy) Bitto Valtellina (Lombardy, Italy) cheeses from Piedmont (Italy) Fontina (Aosta Valley, Italy) Montasio (Friuli Venezia Giulia, Italy) Asiago (Veneto, Italy) Mascarpone (Italy) Camoscio (Italy) Emmenthal (Switzerland) Sbrinz (Switzerland) Gruyere (Switzerland) Emmenthal (French) Brie (French) Commercial cheeses (Netherland) sheep cheeses Pecorino (Tuscany, Sardinia, Lazio, Italy) Roquefort (French) Ricotta (Italy) goat cheeses commercial goat cheese (Italy) Quatre feuilles (French) commercial butters (Italy)
rangec
0.06−0.23 0.06−0.10 0.044−0.086 0.047−0.251 0.046−0.130 0.042−0.106 0.014−0.066 0.022−0.130 0.048−0.138
0.038−0.041 0.025−0.047 0.028−0.030
0.018−0.067
Results are reported as mean ± SD, minimum and maximum for homogeneous groups of samples. Positivity for values ≥0.01%. With respect to the total chromatographic area of fatty acid methyl esters.
a
b
with bacterial acid methyl ester (BAME) mix standard containing cyclopropane fatty acid methyl esters (Sigma-Aldrich, Saint Louis, MO, USA). Content of CPFA was reported as relative percentage response area (%) of the corresponding peak (evidenced in Figure 2) with respect to the total chromatographic area from GC-MS analysis of the other main methyl esters of fatty acids (including C4:0, C6:0, C8:0, C10:1, C10:0, C11:0, C12:1, C12:0, C13:0, C14:0iso, C14:1, C14:0, C15:0iso, C15:0anteiso; C15:1, C15:0, C16:0iso, C16:1, C16:0, C17:0iso, C17:0anteiso, C17:1, C17:0, C18:2, C18:1, C18:0, C18:2cla, C19:1, C19:0, C20:4, C20:3, C20:1, C20:0, C21:0, C22 PUFA, C22:0, C23:0, C24:0). Data Analysis. Data are obtained as duplicate analysis of two independent sample extractions and are presented as mean ± SD of groups of homogeneous samples. The range (minimum and maximum value) of CPFA was also provided. Positivity or negativity of samples to CPFA was defined on the basis of the threshold of 0.01% (CPFA with respect to total chromatographic area of fatty acid methyl esters); this value corresponds to the experimental detection limit, calculated at a signal-to-noise ratio >3. For fermented milk samples, one-way ANOVA and post hoc Tukey-HSD tests at a p level = 0.05 (n = 4) were performed to detect an eventual effect of fermentation and shelf life on CPFA content. All the statistical analyses were performed utilizing SPSS statistical software (Version 20.0, SPSS Inc., Chicago, Illinois, USA).
c
samples of milk from cows fed with forage without maize silages were all negative.9 In this paper, we extend the investigation on the presence of CPFA to about 50 other milk samples, comprising commercial milk samples, bulk raw milk collected from different regions of Italy, countries of the European Union, and China, and milk from different animal species. The content of CPFA detected in cow milk varies greatly according to region of origin, and different trademarks (Table 1). The majority of the cow milk samples collected resulted positive to CPFA, reflecting a large use of ensiled products in cows’ feeding. The collections of milk from Austria and Veneto (Italy) areas show values of CPFA lower than those collected in Emilia Romagna and Lombardy (Brescia, Milan, Reggio Emilia). Among commercial cow milk samples, the two samples negative to CPFA come from mountain regions. Goat, sheep, and yak milks analyzed were all negative to CPFA, indicating that the feeding system is fundamental for the presence of these fatty acids in milk; in fact, these animals are usually fed with pasture grass or, during the winter, with hay. Fermented Milks. CPFA are reported to be present in large amount in cytoplasmic membrane fatty acids of LAB as Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus,15−17 and Lactobacillus casei,18 so it would be expected that they were released in fermented products in significant amounts. To confirm this hypothesis, experimental fermented milks were prepared inoculating different combinations of microorganisms, as reported in Materials and Methods.
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RESULTS AND DISCUSSION Milk Samples. We previously showed that 80 samples of milk from cows fed with maize silage were found positive for the presence of CPFA (0.08 to 0.12%). In contrast, 140 7830
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Figure 2. (a) GC-MS chromatogram of Grana Padano cheese sample fatty acid methyl esters on a SLB5 capillary column and details of the elution zone of CPFA for (b) Grana Padano, (c) milk from Lombardy, and (d) commercial butter. The CPFA peak corresponds to the sum of dihydrosterculic and lactobacillic acid.
to CPFA, while samples of Grana Padano tested are all positive (Table 3). Results obtained for these two cheeses reinforce the hypothesis that the CPFA presence in milk derives from the incorporation in milk triglycerides of the cellular bacterial fatty acids detected in the ensiled products.9 In fact, the product specifications for Parmigiano-Reggiano19 prohibit the use of silages for feeding cows, while the specification rules for Grana Padano20 allow the use of silages. Consequently, the control of CPFA in milk fat could be considered as a quality parameter for cheese productions that prohibit the use of silage feeding. Cheeses coming from mountain regions as Swiss cheeses (Emmenthal, Gruyere) and Aosta Valley (Fontina, Italy) were negative to CPFA, indicating that the use of ensiled forages is infrequent. Goat and sheep cheeses (fresh or seasoned, from several Italian regions) are exempt of CPFA, confirming data reported for milk of these two species. The absence of CPFA in Parmigiano-Reggiano, as well as in other cheeses reported in Table 3, confirms the hypothesis that lactic acid bacteria are not able to release detectable amounts of CPFA in dairy products during manufacturing and seasoning steps. Butter. Among the seven commercial butters analyzed, four were positive to CPFA, showing variable CPFA contents (Table 3), and three were negative. The negative samples were butter declared of alpine origin, butter produced from milk for Parmigiano-Reggiano production, and goat butter, further confirming data reported for goat milk and cheese.
Fermented milk samples were tested for the content of CPFA and compared with the content in the starting milk (Table 2). Results showed that starting bulk milks for yogurt/fermented milk productions were positive to CPFA, containing mean percentages of CPFA of 0.041%, with no significant differences (ANOVA test, p > 0.05) between milk collected in summer (0.043%) and milk collected in winter (0.038%). Fermented milks and yogurt maintained the percentages of CPFA present in the starting milk; in fact, the CPFA contents of fermented products are not significantly different (Tukey multiple comparisons, p level 0.05, n = 4) from starting milk, in all the experiments conducted with different microorganisms. Moreover, CPFA content does not change during the shelf life of the products. These results demonstrate that the most common microorganisms tested and utilized for yogurt or fermented milks production (Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactococci) are not able to release significant amounts of CPFA in the medium and to incorporate them in triglycerides. Therefore, we can conclude that the amount of CPFA found in fermented milks derives only from their initial content in milk and not from the fermentation process. Cheeses. Many different kinds of cheeses were analyzed, focalizing the attention on two important Italian PDO hard cheeses, i.e., Parmigiano-Reggiano and Grana Padano. The results showed that all the samples of Parmigiano-Reggiano (both commercial grated products and samples certified from Consorzio del Formaggio Parmigiano-Reggiano) are negative 7831
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(12) Stevenson, D.; Muck, R.; Shinners, K.; Weimer, P. Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover. Appl. Microbiol. Biotechnol. 2006, 71, 329−338. (13) Rossi, F.; Dellaglio, F. Quality of silages from Italian farms as attested by number and identity of microbial indicators. J. Appl. Microbiol. 2007, 103, 1707−1715. (14) Brusetti, L.; Borin, S.; Mora, D.; Rizzi, A.; Raddadi, N.; Sorlini, C.; Daffonchio, D. Usefulness of length heterogeneity-PCRfor monitoring lactic acid bacteria succession during maize ensiling. FEMS Microbiol. Ecol. 2006, 56, 154−164. (15) Rizzo, A. F.; Korkeala, H.; Mononen, I. Gas chromatography analysis of cellular fatty acids and neutral monosaccharides in the identification of lactobacilli. Appl. Environ. Microbiol. 1987, 53, 2883− 2888. (16) Guillot, A.; Obis, D.; Mistou, M. Y. Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress. Int. J. Food Microbiol. 2000, 55, 47− 51. (17) Béal, C.; Fonseca, F.; Corrieu, G. Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition. J. Dairy Res. 2001, 84, 2347−2356. (18) Broadbent, J. R.; Larsen, R. L.; Deibel, V.; Steele, J. L. Physiological and Transcriptional Response of Lactobacillus casei ATCC 334 to Acid Stress. J. Bacteriol. 2010, 5, 2445−2458. (19) Regulation (EU) N. 794/2011. (20) Regulation (EU) N. 584/2011.
Due to the limited number of butter samples analyzed, further studies are needed to confirm these results. Summarizing, our results suggest that the presence of CPFA in milk and dairy products is derived from their presence in silage forages, where CPFA can be released by bacteria during silage fermentation conditions. On the contrary, lactic acid bacteria, ubiquitous in fermented milk and cheeses, seem not to be able to release CPFA in the conditions of milk fermentation. Therefore, CPFA can be considered interesting molecular markers, able to distinguish milk from dairy cows fed with silage-based diet from milk from cows fed with hay-based diets. The analysis of CPFA can be a useful tool of the quality control for PDO cheeses, such as Parmigiano-Reggiano, whose specifications of production do not allow the use of silages, as well as a marker to differentiate high quality dairy products such as milk, cheese, and yogurt from alpine regions, where cattle feeding is based only on pasture and hay. Further analytical work on feed is in progress to confirm the presence of CPFA in all ensiled feeds.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +39 0521 905407. Fax: +39 0521 905472. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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