ORIGINAL ARTICLE

Inclusion of Pichia guilliermondii on different culture media, on in vitro fermentation of Cynodon nlemfuensis

Inclusión de Pichia guilliermondii en diferentes medios de cultivos en la fermentación in vitro de Cynodon nlemfuensis

Yoandra Marrero, Dailyn Sosa, R. Rodríguez, Yaneysi García

ABSTRACT

The objective of this study was to evaluate the effect of inclusion of Pichia guilliermondii on different culture media on in vitro fermentation of Cynodon nlemfuensis. In vitro gas production technique was used, as well as a completely randomized design, with 6 x 7 factorial arrangement (six treatments per seven hours of fermentation). A strain of P. guilliermondii (Levica 27) was included on one of the treatments, which was cultured in malt extract broth and extract of yeast-peptone-glucose media. Two of the treatments included the supernatant of each medium after centrifugation, and another treatment suspended again cell pellet in a buffer medium. A control without yeast was also included. An amount of 1 g of Cynodon nlemfuensis was incubated as fibrous substrate. Gas production was measured at 2, 4, 8, 12, 16, 20  and 24 h. Results showed that Levica 27 strain, cultured in malt extract broth and extract of yeast-peptone-glucose media, stimulated C. nlemfuensis gas production in a higher proportion (P < 0.001) than when it was cultured in a malt extract broth medium, which demonstrated the influence of metabolites that produce yeasts and their stimulating effect.

RESUMEN

El objetivo de este trabajo fue estudiar el efecto de la inclusión de Pichia guilliermondii en diferentes medios de cultivo en la fermentación in vitro de Cynodon nlemfuensis. Se utilizó la técnica de producción de gas in vitro y un diseño completamente aleatorizado, con arreglo factorial 6 x 7 (seis tratamientos x siete horas de fermentación). En uno de los tratamientos se incluyó la cepa  de P. guilliermondii  (Levica 27), cultivada en los medios Caldo Extracto de Malta y Extracto de levadura-Peptona-Glucosa, en dos de ellos se incluyó el sobrenadante de cada medio después de centrifugación y en otro, se resuspendió el pellet de las células en medio buffer. Se incluyó además, un control sin levadura. Se incubó 1 g de Cynodon  nlemfuensis  como sustrato fibroso. La producción de gas se midió a las 2, 4, 8, 12, 16, 20  y 24 h. Los resultados mostraron que la cepa Levica 27, cultivada en los medios Caldo Extracto de Malta y Extracto de levadura-Peptona-Glucosa, estimuló la producción de gas de C. nlemfuensis en mayor proporción (P < 0.001) que cuando se cultivó en medio Caldo Extracto de Malta, lo que corroboró la influencia de los metabolitos que producen las levaduras y su efecto estimulador.

INTRODUCTION

The use of microbial additives in feed for ruminants, especially yeast, contributes to a better use of food, allowing the increase of productive yields and, consequently, milk and meat availability for humans (Stella et al. 2007, Doležal et al. 2011). Despite the advantages offered by these products, they are not produced in Cuba. Their high prices on the international market make them unaffordable for their livestock use.

Under these conditions, studies were conducted to obtain microbial additives that respond to the specific problems of a Cuban livestock, which is based on fibrous feeds. Currently, promising results have been obtained, in which strains not belonging to S. cerevisiae showed use potential as activators of ruminal fermentation under in vitro conditions, mainly P. guilliermondii, known as Levica 27 (Marrero et al. 2014). Therefore, in vivo studies were recommended to corroborate these results, for which large-scale culture is necessary.

It is known that the design of the culture medium is one of the most important tasks within biological technology. According to Winkler (1988), raw materials may represent between 30 and 80% of total cost of biotechnological products. In addition, the composition of culture medium has to meet all nutritional requirements of the microorganism.

There are means specifically formulated for yeast growth, such as malt extract broth (MEB) and yeast-peptone-glucose (YPG) extract. In previous studies with the strain of P. guilliermondii (Levica 27), it was demonstrated, by indirect methods of growth determination (biomass and optical density), that there are differences between both culture media at 16 h. The highest concentrations of biomass were obtained for YPG medium, with values of 3.88 mg/mL, whereas there were 2.33 mg/mL with MEB. There was also an increase of pH, when the strain was cultured in the MEB medium (Sosa et al. 2015).

These results indicate that culture medium influences on the nature of metabolites produced by the strain, so it is possible that this may also affect the ruminal fermentation process, when it is included as an additive in animal feeding. Regarding these antecedents, this study was conducted with the objective of studying the effect of culturing P. guilliermondii on different media, in in vitro fermentation of C. nlemfuensis.

MATERIALS AND METHODS

Treatments and experimental design. The technique of in vitro gas production and a completely randomized design with 6 x 7 factorial arrangement (six treatments x seven hours of fermentation) were used. The effect of the inclusion of P. guilliermondii growths on different culture media in the C. nlemfuensis gas production was determined. Moreover, the use of 10 g of DM/animal yeast was evaluated, because it is generally included on diets for ruminants (Rojo et al. 2000, Combellas et al. 2002, Beauchemin et al. 2003).

Preparation of yeast inocula. Pichia guilliermondii (Levica-27) strain was used, which belonged to the Bank of Microorganisms from the Department of Bio-physiological Sciences, from the Institute of Animal Science (Mayabeque, Cuba), and its sequence is registered in Gen Bank, with number JF894143.1.

The strain was activated by two subcultures on Petri dishes with Sabouraud agar at 30 °C and 24 h of incubation. Active strain colonies were taken with an inoculation loop and an inoculum of 24 h, with a concentration of 10⁷ cfu mL^-1, was prepared in YPG medium. The inoculum was centrifuged, the supernatant was removed and the pellet was washed and re-suspended in PBS. Erlenmeyers of 250 mL of capacity were used with 100 mL of each medium under study, which was inoculated with microbial suspension at a rate of 1/10 (v/v) and incubated for 16 h on orbital shaker at 30 °C and agitation speed of 110 rpm. After removing these cultures from the shaker, they had a concentration of about 6.10⁷ cfu•mL-1 and pH of 5.74 and 7.03 for YPG and MEB, respectively.

Experimental procedure. Bottles 100 mL of capacity were used, to which 1 g of substrate (C. nlemfuensis) was added, as well as an incubation medium and an inoculum of ruminal microorganisms. These last at a rate of 0.20 of the total volume (16 mL of LRF + 64 mL of incubation medium = 80 mL). The chemical composition of the substrate (% of DM) was 96.21; 4.50; 34.65; 7.01; 0.46 and 0.40 for DM, CP, CF, ash, calcium and phosphorus. It was determined in LASAICA, according to AOAC (2012).

Twelve bottles per treatment and four bottles without substratum were incubated as target, for a total of 40 bottles. Ruminal content of an adult bovine, cannulated in the rumen and fed a diet similar to the evaluated one, was used as inoculum. Ruminal content was removed before feed supply and stored in closed thermos until getting to the lab, where it was filtered through several layers of gauze to form the inoculum.

Bottles were sealed and incubated in a water bath with controlled temperature, at 39 °C. Gas production was measured with a manometer HD8804, coupled to a pressure calibrator TP804 (DELTA OHM, Italy). Pressure readings became volume using a pre-established linear regression equation (gas (mL) = (pressure [103Pa] +4.95)/2.5858); n=132; r=0.991). Gas volume was expressed per gram of incubated DM. Gas production was measured at 2, 4, 8, 12, 16, 20 and 24 h.

Mathematical analysis. In order to analyze the results of accumulated gas production, a curve modeling was conducted by Gompertz model, using a non-linear regression analysis with SPSS 15.0.1 (IBM Corporation 2006). Determination coefficient, significance of fit, parameters and normality of residues: PG (a,b,c,t) = A exp(-be^-ct), were taken into consideration as statistical criteria of model fit goodness.

Homogeneity test of curves was performed according to the methodology presented by Jay et al. (2012a, b). As selection test, probability of null hypothesis fulfillment was used by the corrected Akaike information criterion (AICc). Multiple comparisons were performed, using the root of mean square distance among curves as measure. The Least Significant Difference (LSD) was used as test, with a significance level of P < 0.05. Experimental results were performed with InfoStat statistical package (Di Rienzo et al. 2001).

RESULTS AND DISCUSSION

After the fourth hour, treatments using the yeast and its metabolites showed superior gas production to the control up to the 20th hour. Treatment that cultivated cells of the strain (pellet), re-suspended in a buffer solution, did not stimulate substratum gas production. Nevertheless, it is important to point out that at the 24th hour, gas production of treatments with MEB and YPG supernatant, was equal to that obtained with control, so it stimulated fermentation up to around 20 h.

Results of multiple comparisons of values of accumulated gas production (table 1) show that inclusion of Levica 27, cultivated in YPG medium and its supernatant of its culture in a MEB medium, stimulated gas production of star grass, being higher with the presence of the first. This indicates that cells of Levica 27, as metabolites produced by this yeast in a YPG medium, were used by ruminal microorganisms and increased their fermentative activity in the grass used as substratum, while yeast cells grown in a MEB medium, decreased gas production.

These results agree with studies performed by Marrero et al. (2006, 2010), with a strain of S. cerevisiae under Cuban and Colombian conditions, respectively. In both studies, it was concluded that yeast living cells and produced metabolites, have stimulating effect on populations of cellulolytic bacteria and, hence, the production of short-chain fatty acids (SCFA). This must be taken into account for obtaining ruminal fermentation activator products. Holtshausen and Beauchemin (2010) reported that most of the stimulating activity of these microorganisms is associated to live cells, although, in this study, Levica 27 cells, cultivated in a MEB medium, did not show this effect.

Literature refers to diverse action mechanisms of yeasts as activators of ruminal fermentation. Sirisan et al. (2013) stated that positive effect of yeasts on the rumen is a result of the stimulation of growth of certain populations of microorganisms, with its consequent effect on feed fermentation.

Several authors refer to the stimulating action of S. cerevisiae by providing, by the yeast, growth factors, organic acids, vitamins and small peptides that stimulate bacterial growth. These authors also stated the ability of yeasts to balance redox potential of rumen liquor to create optimal fermentation conditions by bacteria within this organ (Jouany 2001).

Recently, Yuan et al. (2015) proposed that yeast removes the oxygen present in the rumen and promotes the development of anaerobic microorganisms. Therefore, it improves fiber digestion. However, Salvati et al. (2015) found stimulating effects of Saccharomyces cells, living and dead, on feeding of lactating cows, which do not reaffirm this theory.

Mendes et al. (2012) suggested that yeasts are capable of degrading simple carbohydrates, which could favor growth of populations, and also contribute to ruminal pH regulation. Finally, another less studied aspect refers that different species of yeasts are able of producing and storing sugars as trehalose, from 10% to 20% of its dry weight, under unfavorable environmental conditions (Elbein et al. 2003).

 It is known that structure and physicochemical properties of trehalose confer great stability, and this carbohydrate is accumulated in the cytosol of yeasts under abiotic stress conditions, due to its protective effect against dryness, high temperatures, freezing, high salinity and oxidation. Thanks to these properties, trehalose has important applications in the food, cosmetic, and pharmaceutical industry, as well as for research, as indicated by González et al. (2015). These authors report that Candida yeasts were high producers of this disaccharide under thermal stress conditions.

In this study, difference in composition of media that cultured this yeast (MEB and YPG) lies, mainly, in that the former includes malt extract. This component constitutes a diverse energy source, which surely influenced on metabolic processes developed by the yeast strain and final products of these processes that expressed through the pH found in the culture in both media (5.74 and 7.03, respectively). These products of yeast growth also influenced on gas production of C. nlemfuensis, so it is necessary to deepen on their nature.

It is concluded that Levica 27 strain, cultivated in YPG medium, stimulated the gas production of C. nlemfuensis at a higher proportion than within the MEB culture medium, which confirmed the influence of metabolites produced by the yeast and its stimulating effect. Further studies are recommended to explain the nature of metabolites produced by Levica 27 in YPD and MEB media because they stimulated gas production of C. nlemfuensis.

REFERENCES

AOAC 2012. Official Methods of Analysis of AOAC International. 19th ed., Gaithersburg, Md.: AOAC International, ISBN: 978-0-935584-83-7, Available: <http://www.amazon.com/Official-Methods-Analysis-OFFICIAL-ANALYSIS/dp/0935584838/ref=pd_sim_sbs_14_1?ie=UTF8&dpID=31iikC-xl2L&dpSrc=sims&preST=_AC_UL160_SR160%2C160_&refRID=101AB94246X0EM9N7XMW>, [Consulted: April 1, 2016].

Beauchemin, K. A., Yang, W. Z., Morgavi, D. P., Ghorbani, G. R., Kautz, W. & Leedle, J. A. Z. 2003. “Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood chemistry, and subclinical ruminal acidosis in feedlot cattle”. Journal of Animal Science, 81(6): 1628–1640, ISSN: 1525-3163, DOI: 10.2527/2003.8161628x.

Combellas, J., Jacqueline, S., Tesorero, M. & Gabaldón, L. 2002. “Animal response of yearling cattle to the addition of yeast culture to a diet of pasture, poultry litter and wheat middlings”. Zootecnia Tropical, 20(3), ISSN: 0798-7269, Available: <https://tspace.library.utoronto.ca/handle/1807/21217>, [Consulted: September 9, 2016].

Di Rienzo, J. A., Casanoves, F., Balzarini, M. G., González, L., Tablada, M. & Robledo, C. W. 2001. InfoStat. version 2001, [Windows], Universidad Nacional de Córdoba, Argentina: Grupo InfoStat, Available: <http://www.infostat.com.ar/> .

Doležal, P., Dvořáček, J., Doležal, J., Čermáková, J., Zeman, L. & Szwedziak, K. 2011. “Effect of feeding yeast culture on ruminal fermentation and blood indicators of Holstein dairy cows”. Acta Veterinaria Brno, 80(2): 139–145, ISSN: 0001-7213, 1801-7576, DOI: 10.2754/avb201180020139.

González, H. J. C., Alcántar, C. M. A. & Cortés, R. C. 2015. “Producción de trehalosa a partir de levaduras no-convencionales”. Revista Mexicana de Ingeniería Química, 14(1): 11–23, ISSN: 1665-2738.

Holtshausen, L. & Beauchemin, K. A. 2010. “Supplementing Barley-Based Dairy Cow Diets with Saccharomyces cerevisiae”. The Professional Animal Scientist, 26(3): 285–289, ISSN: 1080-7446, DOI: 10.15232/S1080-7446(15)30595-7.

IBM Corporation 2006. IBM SPSS Statistics. version 15.0.1, [Windows], Multiplataforma, U.S: IBM Corporation, Available: <http://www.ibm.com> .

Jay, O., Torres, V., Marrero, Y. & Torres, J. P. 2012a. “Sensibility analysis of homogeneity tests of in vitro gas production curves by Monte Carlo simulation”. Cuban Journal of Agricultural Science, 46(1): 15–22, ISSN: 2079-3480.

Jay, O., Torres, V., Marrero, Y. & Torres, P. 2012b. “Tests assessment for multiple comparisons of in vitro gas curves, from the root of the mean square distance”. Cuban Journal of Agricultural Science, 46(2): 133–138, ISSN: 2079-3480.

Jouany, J. P. 2001. “A new look at yeast cultures as probiotics for ruminants”. Feed Mix, 9(6): 17–19, ISSN: 0928-124X.

Marrero, Y., Castillo, Y., Ruiz, O., Burrola, E. & Angulo, C. 2015. “Feeding of yeast (Candida spp.) improves in vitro ruminal fermentation of fibrous substrates”. Journal of Integrative Agriculture, 14(3): 514–519, ISSN: 2095-3119, DOI: 10.1016/S2095-3119(14)60830-3.

Marrero, Y., Galindo, J., Aldama, A. I., Moreira, O. & Cueto, M. 2006. “In vitro effect of Saccharomyces cerevisiae on the microbial rumen population and fermentative indicators”. Cuban Journal of Agricultural Science, 40(3): 313–320, ISSN: 2079-3480.

Marrero, Y., Ruiz, O., Corrales, A., Jay, O., Galindo, J., Castillo, Y. & Madera, N. 2014. “In vitro gas production of fibrous substrates with the inclusion of yeast”. Cuban Journal of Agricultural Science, 48(2): 119–123, ISSN: 2079-3480.

Mendes,  de A. P. N., Robson, D. E., Oliveira, A. F., Silva, F. C. E., Castro, G. L. & Rosa, C. A. 2012. “Aerobic fungi in the rumen fluid from dairy cattle fed different sources of forage”. Revista Brasileira de Zootecnia, 41(11): 2336–2342, ISSN: 1806-9290, DOI: 10.1590/S1516-35982012001100006.

Rojo, R., Mendoza, M., García, B., Bárcena, G. & Aranda, I. 2000. “Intake and digestibility of tropical grasses in steers with nitrogen supplementation and Saccharomyces cerevisiae”. Revista de la Facultad de Agronomía, 17(4): 358–370, ISSN: 0378-7818.

Salvati, G. G. S., Morais Júnior, N. N., Melo, A. C. S., Vilela, R. R., Cardoso, F. F., Aronovich, M., Pereira, R. A. N. & Pereira, M. N. 2015. “Response of lactating cows to live yeast supplementation during summer”. Journal of Dairy Science, 98(6): 4062–4073, ISSN: 0022-0302, DOI: 10.3168/jds.2014-9215.

Sirisan, V., Pattarajinda, V., Vichitphan, K. & Leesing, R. 2013. “Isolation, identification and growth determination of lactic acid-utilizing yeasts from the ruminal fluid of dairy cattle”. Letters in Applied Microbiology, 57(2): 102–107, ISSN: 0266-8254, DOI: 10.1111/lam.12078.

Sosa, D., García, Y., Marrero, Y., Albelo, N. & Moreira, B. 2015. “Characterization of Pichia guilliermondii (Levica-27) for use as microbial additive in animal production”. In: II Seminario Internacional de Sanidad Agropecuaria, Centro de Convenciones Plaza América, Varadero, Cuba: Centro Nacional de Sanidad Agropecuaria (CENSA), p. 297, ISBN: 978-959-7125-45-7, Available: <http://www.sanidadagropecuaria.com/>, [Consulted: September 9, 2016].

Stella, A. V., Paratte, R., Valnegri, L., Cigalino, G., Soncini, G., Chevaux, E., Dell’Orto, V. & Savoini, G. 2007. “Effect of administration of live Saccharomyces cerevisiae on milk production, milk composition, blood metabolites, and faecal flora in early lactating dairy goats”. Small Ruminant Research, 67(1): 7–13, ISSN: 0921-4488, DOI: 10.1016/j.smallrumres.2005.08.024.

Winkler, M. A. 1988. “Optimisation and time-profiling in fermentation processes”. Progress in industrial microbiology, 25: 91–150, ISSN: 0079-6352.

Yuan, K., Liang, T., Muckey, M. B., Mendonça, L. G. D., Hulbert, L. E., Elrod, C. C. & Bradford, B. J. 2015. “Yeast product supplementation modulated feeding behavior and metabolism in transition dairy cows”. Journal of Dairy Science, 98(1): 532–540, ISSN: 0022-0302, DOI: 10.3168/jds.2014-8468.

Received: 21/6/2016
Accepted: 07/9/2016

Yoandra Marrero, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email: ymarrero@ica.co.cu