ORIGINAL ARTICLE

Effect of the use of fermentation promoters with or without probiotics on the profile of fatty acids, amino acids and cholesterol of milk from grazing cows

Efecto de la utilización de los promotores de la fermentación, con probióticos o sin ellos, en el perfil de ácidos grasos, aminoácidos y colesterol de la leche de vacas en pastoreo

M.A. Galina,^I A. Elías,^II P. Vázquez,^III J. Pineda,^IV B. López,^I M.A.Velázquez,^I

ABSTRACT

This study reviews the development and use of fermentation promoters and probiotics on the profile of fatty acids, amino acids and cholesterol of milk from grazing cows. A herd of 35 cows (511 ± 12 kg), which were in the middle of lactation, Zebu crossings, over a silvopastoral system with star grass (Cynodon plectostachyus) and brachiaria (Brachiaria brizantha), and browsing legumes, with 3 kg of fermented promoters (FP), were provided with 1.5 kg of lactobacilli per day as supplement (LAB). The grazing area was 20.9 ha. Another herd of 28 animals (514 ±14 kg), grazing on 18.5 ha, supplemented with 6 kg/d of a commercial concentrate with 160 g of commercial probioric (CP), was used. An amount of 8 commercial milks were sampled. The milk from the three treatments was weighed each week. The average of production was 17 kg for LAB, of 14 kg/d in silvopastoril system (SP) and 16 kg in CP (P<0.05). The saturated fatty acids and unsaturated fatty acids showed differences in the three treatments (P<0.05). Polyunsaturated fatty acids, omega 3 and conjugated linoleic acid were 34%, 46% and 68% higher in CP, silvopastoral system and LAB compared to commercial milk (CM). Results have demonstrated that grazing diverse green fresh forages improve milk quality due to the increase of unsaturated fatty acids. The LAB allowed a decrease of biohydrogenation. LAB and SP provided the milks with highest amount of omega 3.

Key words: probiotic, quality, milk, cows, tropic.

RESUMEN

Palabras clave: probiótico, calidad, leche, vacas, trópico.

INTRODUCTION

The original studies on management of ruminal fermentation were carried out by Cuban researchers (Elías, 1971), with a posterior large revision on its use (Elías 1983). Later, several studies were published in Mexico, about the effect of FP on different species and grazing systems, with an increase on the use of cellulose of fiber forages due to a significant increase on cellulolitic bacteria population (Galina et al. 2000, 2002, 2003, Galina et al. 2004abc, Ortíz et al. 2001, 2002 and Puga et al. 2001abc).On the other hand, the knowledge about the importance of nitrogen degradation, by ruminal microorganisms, has allowed a rational inclusion of urea on diets, plus mechanical or chemical treatments of forages that improve their digestibility, with the use of FP and LAB (Galindo and Marrero 2005, Ortiz et al. 2007, Gutiérrez et al. 2012ab).  The formation of microbial intestinal digestible protein (PDIM) may reach 80% or more in diets containing an abundant production of ruminal microorganisms in formulas supplemented with non-protein nitrogen sources, while with commercial concentrates, most of the intestinal digestible protein comes from food (PDIA). Therefore, several techniques have been used for protecting protein against the action of ruminal microorganisms (Elías 1983).

Grasses and some legumes provide the base for animal feeding in tropical husbandry. It is characterized by a group of genera and species, and by their wide adaptation to different environments, known as “plasticity” (Peters et al. 2010 and Tittonellet al. 2010).It is possible to use these forages efficiently when bacterial populations of rumen cover the energy requirements, essential nitrogen constituents, minerals and other nutrients (Elías 1983). Otherwise, it could reduce its intake and usage, which could be corrected with the use of activators of ruminal fermentation that increase digestive efficiency (Puga et al. 2001abc). The responses to the use of microbial activators, which are more frequently repeated in these studies, are associated to the production of volatile fatty acids (VFA), modification of ruminal pH and increase of bacteria that are responsible for fiber degradation (Lila et al. 2004).

During the last years, the Institute of Animal Science (ICA) from Cuba has developed a biologically active product called VITAFERT, enriched with yeasts and lactobacilli, organic acids of short carbonated chains, trace elements and a low pH (Elías and Herrera 2008 and Gutierrez et al. 2012ab). This product has been used as microbial additive in pigs and calves, to prevent or decrease diarrheas, as growth stimulant for non-ruminant animals, and in the ruminal fermentation of cattle (Gutiérrez 2005).

From the first decade of this century, modifications on the use of lactic bacteria in ruminants were added to the original works on ruminal fermentation (Elías and Herrera, 2008). This demonstrated its effectiveness on digestibility of fibrous forages (Galina et al. 2007ab and Gutiérrez et al. 2012ab). The results of all these researches have had a significantly higher effect on fiber degradation in the rumen that has allowed the development of alternative management systems with or without LAB supplementation to improve product quality (Gutierrez et al. 2012a).

On the other hand, studies on the profile of polyunsaturated fatty acids (PUFAs), mainly linoleic acid (LA, C18.3 cis-9, cis-12) and conjugated alpha linoleic acid (CLA, C18:3 cis-9, cis-12, cis-15), have demonstrated that they can be found, in high proportions, in lipids of forage and of some supplements (Shen 2011, Castillo et al. 2013, Zened et al. 2013 and Rubino 2014). These acids are part of the diet of ruminants and, depending on their concentration, modify the profile of fatty acids from milk and meat. Their composition is characterized by having a higher volume of unsaturated fatty acids than of saturated ones, which increase their saturation because of the process of BH in the rumen (Castillo et al. 2013). Several factors that affect the process of BH from LA and CLA have been studies, as well as nutritional strategies showing positive results in the increase of trans-vaccenic acid (C18:2 trans-11, TVA) and conjugated linoleic acid (Cis18:2 cis-9, trans-11, CLA) in milk (Galina et al. 2009a,  2010, 2012). These compounds have potential benefit effects to human health (Harfoot and Hazlewood 1997, O´sheaet al. 1998, Herrera et al. 2004 and Khanal 2004).  

The objective of this study was to revise the progress in the management of ruminal fermentation, particularly evaluate the effect of LAB supplement on milk production and its profile of essential fatty acids in animals grazing and browsing in a mixed system of grasslands and tropical forest, with or without the use of RF, alone or together with LAB, compared to commercial concentrates supplementation (COM).

MATERIALS AND METHODS

The study was carried out in“El Fresno” farm, Suchitlán, Colima, at 19°23’ N, 103°41’ W, and 1,400 m o.s.l. According to Köppen, the climate is classified as Aw1 (w), with rains from July to October, 1,000 mm per year. Dry period lasts from 8 to 9 months, with an average temperature of 25°C.

A herd of 35 milking cows was used, which were in the middle of lactation(511 ± 12 kg), and Zebu crossed. The animals were grazing in a silvopastoral system (SP) since July, which contained a mixture of tropical grasses from “zacates”: star grass (Cynodon plectostachyus) and brachiaria (Brachiaria brizantha), with browsing of legumes in tropical forest, supplemented with 3 kg of ruminal fermentation agent, with or without the addition of 1.5 kg/day of a probiotic of lactic bacteria (LAB) as a supplement during the silvopastoral period. The total grazing area was 20.9 ha, with a mixture of tropical grasses from “zacates”: star grass (Cynodon plectostachyus) and brachiaria (Brachiaria brizantha), accompanied by browsing of legumes in tropical forest. The browsed tropical forest included Mimosa pudica, Plumera rubra, Bunchosia palmeri, Cordia alliodora, C. dentata, Platymiscium fasiocarpum, Erythroxylum mexicanum, E. rotundifolium, Caesalpina plumeria, Guttarda elliptica, Randiaca pitata, Caesalpina coriaria and Desmodium spp. The stocking rate was between 3.6 and 5.9 AU/ha.

At the same time, a second herd of 28 animals (514 ±14 kg) was used, which were grazing in 16.5 ha of a silvopastoral system, supplemented with 6 kg of a commercial concentrate per milking cow of 160 g of CP (COM). The milk of the three treatments was weighed individually each week during the observation. Samples were taken, every week, from the milk of each group to measure fatty acids. 

During the study, the forage exceeded the capacity of voluntary intake of lactation cows. The supplementation with probiotics (LAB) contained around 4 x 10⁷cfu of lactic bacteria, composed by Lactobacilos plantarum, L. delbrueckii, L. helvaticus; Lactoccocus lactis, Leuconostoc mesenteroides, and Bifidus spp. over a mixture of 35% molasses and 65% cheese serum. The fermentation promoter (3kg/d) contained a mixture of molasses (18%), cotton meal (16%), rice skin (10%), maize (14%), poultry manure (10%), fish meal (8%), beef fat (5%), salt (4%), lime, calcium carbonate (3%), cement (1%),  mineral salts (2%),  calcium orthophosphate (2%), urea (5%) and ammonia sulfate (2%). The volumes of ingested dry matter were calculated per each cow, taking representative samples in grazing, based on the energy and protein needs for maintenance, growth, milk production and physiological state, according to the methodology, which uses the system of milk forage units. (Jarrige 1995)

The analysis of fatty acid methyl esters (FAME) was performed by separate extraction, using gas chromatography (Varian model 3800), equipped with an automatic sampling (CP 8410) and a FID detector. The chromatographer has a capillary column of fused silica (60 m, 0.25 mm (id) 0.25 micra; DB 23 film, J and W Supelco). FAME peaks were identified by comparison with retention times, with a known mixture of fatty acid standards (Sigma-Aldrich).

The volatile compounds were determined using the modified technique of dynamic headspace. Samples were purged by bubbling helium and extraction was carried out for 60 min with helium at a rate of 50 mL/min. Volatile components were absorbed into a glass trap, filled with 0.20 mg of Tenax TA, 60/80 sieve and 0.05 mg of Carbopack C, 40/60 sieve. Thermal desorption was performed by a heating trap at 220 °C for 5 minutes with a flow of helium carrier gas (50 mL/min) in an automatic system of thermal desorption (TDS2, Gerstel GmbH).The gas analysis was performed with a chromatographer, model Agilent 6890 GC N, connected to a detector of selective quadrupole mass (TME),model 5973. A capillary column of fused silica, covered with dimethyl polysiloxane (HP 1, Agilent Technologies, USA) with 30 m, 0.32 mm (id), 0.25 micra of film thickness, was used to analyze the volatile profile of milk. Operating conditions were in a helium flow of 1.2 mL/min, the transfer line to the ME at 250 °C of splitless open interface. Thermal desorption was performed at 220 °C for 5 minutes with a flow of helium carrier gas (50 mL/min), in an automatic thermal desorption system (TDS2, Gerstel GmbH).Operating conditions were in a helium flow of 1.2 mL/min, the transfer line to the ME at 250 °C of splitless open interface. The temperature of the program was 10 min at 40 °C, with heating speed of 10 °C/min up to a peak of calibrated 150 °C for 12 min. The scanned mass spectrometer ranged from 29m/z to 400m/z in 0.5 s of cycle time. The ion source was set at 230 °C and spectra were obtained by electron impact (70 eV). The detected volatile compounds were identified by the study of DM spectra, compared to data of Wiley (Wiley and Son, Germany).Each sample was analyzed in duplicate. Profiles of volatile fatty acids in milk were expressed as percentages. The chromatographer performs an automatic sampling (CP 8410) with FID detector.The chromatographer has a capillary column of fused silica (60 m, 0.25 mm (id) 0.25 micra with DB 23 film, J and W,Supelco). FAME peaks were identified by comparison with retention times, with those of a known mixture of fatty acid standards (Sigma-Aldrich). The standard CLA (cis-9, trans-10, cis-12 3%) was obtained from Larodan (Malmö, Sweden).

Yij = μ+ti+Ej

Yij= values of fatty acids             i=1,2..., 4      j=1,2...,8

μ= general mean

ti= effect of the i-th treatment

Ej= effect of random error.

RESULTS

The average of milk in both groups was 17.5, (LAB); 14.1 (SP) and 16.5 (COM) kg/d (P<0.05).The feeding system affected significantly the profile of fatty acids in the milk of the studied cows. This effect was measured by the content of fatty acids from feeding systems, in percentages of saturated and unsaturated LAB 66.17:34.04%; SP 65.82:34.23%; COM 67.97:32.30%; CM 67.77:32.35%, as table 1 shows. The content of polyunsaturated fatty acids, omega-3 was LAB 0.51%, SP 0.33% and COM 0.27% superior to those of commercial milk (CM), which had 0.18% of average. Results of omega 6 were 1.77 % LAB, 1.59 % SP and 1.50% COM, while commercial milk had an average of 1.47% (P<0.05). The relation omega 6/omega 3 was 3.47:1 LAB; 4.82:1 SP; 5.16 COM and 8.17 commercial.

Regarding saturated fatty acids, CM and COM were superior to the milk of grazing animals, which showed no differences among them (P>0.05). Unsaturated fatty acids had a similar performance but LAB and SP were superior to COM and CM (P>0.05).Differences among the four milks were found in the percentage of polyunsaturated fatty acids, being intermedia LAB the highest for SP and COM and the lowest for CM (P>0.05). The analysis of variance showed that the feeding system only modified the concentration of polyunsaturated fatty acids (P<0.01). However, monounsaturated fatty acids were only affected (P<0.01) between LAB and SP compared to COM and CM. Regarding polyunsaturated fatty acids, LAB had the highest (P<0.05) percentage, superior to the remaining types of milk. There was significant effect (P<0.01) of feeding system over the different types of milk.

Table 3 presents a summary of the content of saturated, monosaturated, and polyunsaturated fatty acids. There is a higher percentage of saturated in COM and CM compared to LAB and SAP. However, it is particularly important the difference in the content of omega 3, being higher in milks of pastors, especially in those supplemented with probiotics. After analyzing the cholesterol content in different types of samples, LAB recorded a content of 83.2 mg/100 mL and SP registered 84.4 mg/100mL, showing significant differences with COM (87.5 mg/100 mL) and CM(89.1 mg/100 mL). In this regard, the feeding system, with or without probiotics, showed lower amount of cholesterol in both treatments.

An important element is the relation omega 6/omega 3, which, in this observation, was 3.47 for LAB and 4.82 for SP, slightly within a range lower than 5, while the same silvopastoral system, when supplemented with commercial concentrate, has a relation from 5.56 to 1, probably blocking the beneficial effect of omega 3, and the average of commercial milk went from 8.17 to 1. This was superior to the limits that allow a beneficial use for humans.

DISCUSSION

The performance of ingestion and rumination has been widely documented, regarding the nature of diet, essentially, its superior plant maturity and provided fitness. These are aspects that could on influence the filling of organs and dry matter degradation rate in the digestive tract (Van Soest 1982).The fermentation promoters (FP) have demonstrated to have the elements that allow a better utilization of cell walls due to several factors including the presence of a source of soluble carbohydrates to offer energy as ATP of anaerobic bacteria (Galina et al.2003).

By analyzing the ruminal liquor, the pH values obtained with the use of FP were ± 6.9 even when there is no single consensus of pH value where the functioning of ruminal microbiota is optimized. The figures found in all treatments are between the physiological limits of 6.0 and 7.2 (Elías 1971, 1983, Calsamiglia et al. 2002 and Krause and Oetzel 2006), as optimal values to guarantee the digestion of cellulose, and favor the increase of growth rate of cellulitic/hemicellulolitic microorganisms, their enzymatic activity and the products of their metabolism (Marrero 2005). The values of pH reached with the FP have a positive effect exerted by microbial activators to stimulate bacterial growth and contribute to increase the DM intake, mainly NDF (Puga et al. 2001abc).

Castañeda et al. 2010 achieved similar results in the performance of goats and sheep, because they demonstrated an increase of VFAs concentrations from 2 and 8 hours after food ingestion. At the same time, these authors reached a negative correlation (r² = 0.454, P <0.01) between pH and the concentrations of these organic acids. A similar performance is possible with the use of RF/LAB at this level indicates that in the ruminal mixed ecosystem had to be influenced not only by the concentration of these acids, but also by other factors such as the buffering capacity of the medium (Ramos and Antonio 2009 and Galina et al. 2009ab).This should have occurred in these treatments, as a result of increased chewing and rumination, as it was previously stated, which brings about a higher production and segregation of saliva (Gutiérrez et al. 2012ab).The possibility of buffering these substances (carbonates and phosphates), plus urea, and amounts of VFAs, acetic and lactic acids, contained in LAB, and produced in the rumen, together with pH stability, should substantially improve the synthesis of microbial protein (Elías 1983), and, consequently, increase animal response.

According to Smith (1975), it is possible to estimate the ruminal microbial mass from the AGVs concentrations. Studies carried out in Cuba determined the optimum level of 6 mL kg LW^-1 of LAB in the ration, for increased production of microbial biomass (Gutierrez et al.2012ab). This evidences better ruminal fermentation and, consequently, more degradation of forage enriched in cell walls and microbial mass, which becomes part of digesta as bypass protein of excellent amino acid composition, leaves the rumen and is absorbed in the small intestine (López 2009).

Although there are several in vivo studies describing improvements in degradation rate of DM in the diet of cattle consuming basically fiber, with the addition of microbial additives from mixed cultures of yeast and lactobacilli, such as those conducted by Flores (2000) when using 1% of strains of Lactobacillus plantarum in a basic diet containing concentrate and alfalfa, where improvements in degradability were obtained in other studies developed by Castillo (2009) and Gutiérrez et al.(2012ab) evaluated microbial preparations of S. cerevisiae, related to characteristics of ruminal fermentation in cows fed fiber diets, and found an increase of cellulolitic and total viable bacteria. With the use of LAB, there was a significant improvement in the content of essential fatty acids of milk from the animals (Galina et al. 2009 and Elías et al. 2010).  Nevertheless, differences found in the ingestion of DM and NDF with LAB diet, regarding other treatments of this study, can be attributed to similar effects to those previously achieved in cattle, and thus, an increase of disappearance rate of fibrous material in the rumen, similar to that described by Galina et al. (2007).

 The results with LAB in the ration suggest that there are major changes in the ruminal microbial activity, resulting in an increased of fermentation ability of structural carbohydrates, by degrading complex carbonated chains and release simple strings that were used by cellulolytic bacteria, as energy sources for their growth since their beginning, plus the contribution of LAB with peptides and amino acids within its true protein (Elías, 1983 and Galina et al., 2008ab, 2009a). This is demonstrated during the kinetics performance of the curve, where LAB stimulatory activity was observed, perhaps associated to living cells, plus their activity in the ruminal liquor, since the beginning of degradation kinetics and its extension (Gutierrez et al. 2012b).

In studies of Gutierrez et al. (2012ab), with the use of probiotics, the response to the characteristics during degradation kinetics of DM showed that soluble fraction (A) was the same in all treatments, mainly because the incubated fibrous material was the same (B. brizantha hay). This indicator was estimated from the material lost during bag washing, at zero hour, without ruminal incubation. In this regard, it can be stated that the values of potential degradation (A + B) were determined, primarily, by the insoluble, but degradable, fraction (B).This fraction, according to Ortíz et al. (2007)in studies developed with grasses, express the permanence time of this type of feed in the rumen, and it is related to the time of adaptation and colonization of microorganisms to degrade this fraction. At the same time, there is a high degradation rate (c) of insoluble fraction (B) with values of 2.9 h^-1. Similarly, the highest value of effective degradability (ED) of the potentially degradable fraction was determined by the lowest ruminal turnover rate (2% h). In the latter, the effective degradation decreases with the increase of ruminal turnover rate. This confirms the importance of using effective degradation and not the potential one, for calculating diet, as proposed by Leichtle and Cristian (2005).

Regarding the product quality, the minimum content of saturated fatty acids (SFA) was found in the milk of grazing animals, being significantly superior when the probiotic was added. The literature states that a low content of SFA may favor human health because of the information accumulated of the effect of blocking of blood vessels on coronary diseases (Pfeuffer and Schrezenmeir 2000). Results of the present study explain that the feeding system, in general, and specifically free grazing in a silvopastoral system, allows each cow, mainly in forage-diversity areas, to form a diet according to their own needs, which have a good effect on nutritional characteristics of milk, making it favorable for health. The highest content of trans-fatty acids was present in grazing milk. Negative effects of trans-fatty acids on health were considered similar to those reported for saturated fatty acids (Sicchiari, 2008) until recently. Negative effects of trans-fatty acids on coronary pathologies and cytotoxicity were determined from observations on the metabolism of hydrogenated fatty acids, produced during the manufacturing of industrial feeds. Trans derived from processes of ruminal biohydrogenation, like those produced by rumen, have demonstrated positive effects on human health (Wencelová et al. 2015). In the present study, this fact was observed for most of C18:1 transvaccenic acids, though the action of 9 desaturation, where it is metabolized in C18: 2 11 trans 9 cis, which represents one of the most important precursor of beneficial CLA (Castillo et al. 2013). Therefore, after having this relatively new knowledge, the role of trans fatty acids in the feeding system of ruminants in free grazing have to be reevaluated for producing a “better” milk for the health of the consumer. This fact is a great concern in Mexico because a big part of the population suffers from obesity or overweight, which may be translated into degenerative chronic diseases, mainly coronary changes (Rubino 2014).

The beneficial effect of omega 3 and omega 6 polyunsaturated acids have been abundantly documented (Colavilla et al. 2014). New studies have demonstrated the importance of maintaining a rate lower than 5:1 between omega 6 and omega 3, because superior concentrations block the beneficial effects of omega 3, affecting health (Colavilla et al. 2014 y Rubino 2014). Only LAB with 3.48 and SP with 4.79 reached this parameter while grazing supplemented with commercial concentrates is slightly superior (5.54) and the average of 8 commercial milks was 8.40, which means that the little contained omega 3 would have no effect on health because it is blocked by omega 6 (Simopoulos 2002 and Strandvik 2011).

 Even though the differences demonstrated between the two systems, omega 3/omega 6 relation and CLA values were favorable for both systems, which demonstrates the importance of biohydrogenation in milk production, which decreases with the use of lactic bacteria. Significant differences in the profile of beneficial fatty acids from the milk of grazing or stabulated animals, with the addition of a supplement of lactic acid bacteria, compared to commercial milk, demonstrates the importance of biohydrogenation in the ruminal metabolism for milk quality and consumer health (Galina et al. 2013).

Results of BH with LAB were similar to those obtained in diets supplemented with organic acids or plant with oils (Wencelová et al. 2015), which suggests that lactic acid bacteria have a form of ruminal fermentation that may have a similar effect, and results in a better quality of milk (Galina et al. 2012). Therefore, several observations have been performed, comparing feeding system in stabulation or grazing with or without the use of lactic bacteria supplementation. The significant differences in the profile of essential fatty acids among the milks of grazing animals, with the addition of lactic bacteria supplementation, compared to commercial milk, demonstrated the importance of BH in ruminal metabolism for milk quality and consumer health. Then, animals from silvopastoral system with supplementation of PF and probiotics were those that produced significantly better quality milk, compared to grazing without probiotics or those stabulated with or without probiotics.

CONCLUSIONS

Results have demonstrated that the two feeding systems in grazing, even if both are mainly composed of fresh green forages, improve the quality of milk probably due to an increase of USFA in diets. Nevertheless, due to the decrease of BH using LAB, there is a production of better quality milk in their profile of essential fatty acids, and a favorable significant difference was observed, even compared to SP (P ≤ 0.05), which meant that with a decrease of BH due to lactic flora that decreases when there is a substrate with higher diversity of forages, as in the case of animals in LAB.

ACKNOWLEDGEMENTS

The authors would like to thank PAPIIT UNAM IT 202014-3 and Cátedra CONS-207 of FES-Cuautitlán UNAM.

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Received: November 24, 2015
Accepted: May 5, 2016

M.A. Galina, Facultad de Estudios Superiores Cuautitlán Universidad Nacional Autónoma de México. Email: miguelgalina@hotmail.com