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Article

Effect of Live Yeasts (Pichia guilliermondii) on In Vitro Fermentation of Corn Stover as a Fibrous Substrate

by
Bexy González Mora
,
Oscar Ruiz-Barrera
,
Francisco Castillo Rangel
and
Yamicela Castillo-Castillo
*
College of Animal Science and Ecology, Autonomous University of Chihuahua, Chihuahua 31000, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(1), 17; https://doi.org/10.3390/fermentation9010017
Submission received: 27 November 2022 / Revised: 14 December 2022 / Accepted: 16 December 2022 / Published: 24 December 2022
(This article belongs to the Special Issue Rumen Fermentation)
Review Reports Versions Notes

Abstract

:
The objective of the present study was to examine the potential utilization of the yeast strain Pichia guilliermondii (Levica 27) on the ruminal fermentation of corn stover. Two in vitro treatments were evaluated: (A) filtered rumen liquid + powdered corn stover + yeast-free culture medium and (B) filtered rumen liquid + powdered corn stover + culture medium inoculated with yeast. Samples were collected at 0, 6, 12, and 24 h and examined for effects on culture pH, ammonia nitrogen (NH3-N), the fiber digestibility percentages of neutral detergent fiber and acid detergent fiber, the digestibility of dry matter, and the concentration of volatile fatty acids (VFAs). The results showed that the addition of Levica 27 had no effect on the ruminal pH, NH3-N or the digestibility indicators (p > 0.05). However, at 12 h, the inclusion of Levica 27 increased (p < 0.05) the molar concentrations of total VFAs and acetic and propionic acids in ruminal fluid and decreased (p < 0.05) the C2:C3 ratio. It is concluded that Levica 27 seems to have a stimulating effect on ruminal fermentation over a range within 6 and 12 h, a result that should be taken into account when in vivo studies are performed with this yeast strain.

1. Introduction

Since the beginning of the present century, nutritionists have explored the concept of the manipulation of ruminal fermentation [1], with the aim of improving the metabolism of nutrients in the rumen, and as a consequence, increasing the productivity of animals [2,3,4]. The elimination of antibiotic and growth promoters in animal production by the European Union constitutes a challenge for the scientific community, since the aim should be the search of additives that are innocuous for the animals and the consumers. This is the case for direct-fed microbials (DFMs), which are made up of beneficial viable microorganisms that are being studied with increasing scrutiny for new ways to improve the performance and health of ruminant animals [5]. Among these DFMs, yeasts constitute one of the alternatives for improving ruminant production and nutrient use efficiency [6], due to their ability to stimulate better microbial growth in the rumen—mainly populations of cellulolytic bacteria [7], enhancing in vitro and in vivo fiber digestibility [8], stabilizing ruminal pH [9,10], decreasing methane production [10,11,12], improving fermentation patterns [13], and reducing pathogen concentrations, and therefore, increasing meat production [14], milk production (kg/d), and milk protein yield (kg/d) [15]. Moreover, Ruiz et al. (2016) [16] showed that the inclusion of Candida norvegensis in an in vitro fermentation study increased the ruminal fermentation parameters of oat straw as well as microbial growth, with a reduction in methane production. Castillo et al. (2016) [17] also reported significant increases for in vitro gas production when alfalfa hay and oat straw were used as substrates and treated with Candida norvegensis, suggesting that this yeast strain improves ruminal digestion. However, not all the yeast strains tested have been capable of modifying ruminal metabolism or enhancing animal productivity [18,19]. Despite these proven effects of yeast supplementation, the results are variable and inconsistent, as has been reported in previous studies, possibly due to the yeast strain, dose, and mode of application of the yeast, and since there are some strains with specific effects and others exhibiting multifunctional effects [20,21]. Hence, we hypothesize that the addition of Pichia guilliermondii as a microbial enhancer improves ruminal in vitro fermentation using a fibrous substrate. The objective of the current in vitro study was to demonstrate the efficacy of the yeast strain Pichia guilliermondii (Levica 27) on the fermentation of corn stover.

2. Materials and Methods

2.1. Location of the Study

The work was carried out in the Animal Nutrition Laboratory of the Faculty of Animal Science and Ecology of the Autonomous University of Chihuahua (UACH), located in the Periferico Francisco R. Almada, 1.0 km from the city of Chihuahua, Chihuahua, Mexico (latitude 28°35′10.9″ north; longitude 106°6′26.6″ west; altitude 1440 msnm).

2.2. Additives

The yeast strain Pichia guilliermondii Levica 27 was isolated from the Holstein cow ruminal ecosystem and maintained in the Bank of Microorganisms of the Institute of Animal Science (Mayabeque, Cuba) with record number 980 of the World Data Center for Microorganisms (WDCM), and number JF894143.1 in GenBank [22]. Yeast strain samples of P. guilliermondii were inoculated into malt extract broth (DIBICO® Cuautitlán, Izcallí. México) and incubated for 24 h at 30 ± 2 °C, and in an orbital shaker incubator (New Brunswick Model Innova 4000, Nijmegen, Netherlands) at 110 rpm. The probiotic was prepared by placing 50 mL of this inoculum into 450 mL of a medium containing 20 g/L of molasses and 10 g/L of urea [23], and incubated at 30 ± 2°C for 16 h (time of maximum yeast growth). The Levica 27 culture had a final concentration of 1.33 × 108 CFU/mL.

2.3. Experimental Treatments

Two treatments were evaluated: (A) filtered rumen liquid + corn stover substrate + yeast-free culture medium, and (B) filtered rumen liquid + corn stover substrate + culture medium inoculated with yeast; the sampling times were 0, 6, 12, and 24 h. Four repetitions per treatment were performed. The reason for using only corn stover as a substrate to define the effect of live yeasts was that it is a very important ingredient in diets for ruminants, with a rumen-filling purpose. However, it is important to clarify that it is rarely used as the only ingredient in these types of diets.

2.4. Experimental Procedure

The corn stover used as substrate was previously dried in the sun for two days, and finally ground through a 1.0 mm mesh. The chemical composition was determined according to [24] (Table 1).
Subsequently, 0.5 g of substrate was weighed into Ankom® F57 fiber filter bags (ANKOM Technologies, Fairport, New York, NY, USA) with a pore size of 25 μm, and each bag was placed inside the 250 mL flasks that were used for the incubations. Ruminal fluid was extracted from two Angus × Hereford donor cows, with an average weight of 250 kg, which were housed in individual pens and fed for seven days with a corn-stover-based diet prior to taking ruminal fluid. The animals were provided with free access to water. Ruminal fluid extraction was performed through an esophageal tube before the first food offering (9:00 a.m.). The ruminal fluid was filtered through muslin and used to prepare the fermentation medium, which contained ruminal fluid and buffer solution in a 2:1 ratio [25]. Prior to incubation, 120 mL of the fermentation medium was poured into each flask, as well as 30 mL of Levica 27 culture (having approximately 2.5 g of yeast/mL, which is equivalent to 50 g of yeast per animal). The control treatment had fresh inoculated culture medium added [16]. Finally, the 250 mL fermentation flasks were incubated at 39 °C and at 110 rpm in an orbital shaker incubator (New Brunswick Model Innova 4000, Nijmegen, The Netherlands). At each sampling time, four bottles were randomly withdrawn from each treatment. The entire procedure was carried out under anaerobic conditions via bubbling with CO2.

2.5. Analyses

The pH was measured with a table pH meter (Hannah Instruments, Model HI 9017, Arvore-Vila do Conde, Portugal), and NH3-N was determined according to the methodology of [26] via UV spectrophotometry (Varioskan Flash, Thermo Scientific, Vantaa, Finland). The molar concentrations of volatile fatty acids (VFAs) were obtained via gas chromatography with flame ionization detection using a Claurus 400® gas chromatograph (Perkin Elmer, Shelton, CT, USA) with a Varian capillary CP-wax58 (FFAP) CB column (15 m × 0.53 mm, 0.5 µm). The initial temperature of the oven was 115 °C, which was maintained for one minute, then increased by 10 °C per minute until a temperature of 190 °C was reached. The carrier gas was helium at a constant pressure of 3 psi [27]. The injected volume of the sample was 0.6 μL, which was previously conditioned with meta-phosphoric acid according to the procedure described by [28]. The percentages of neutral detergent fiber (NDF) and acid detergent fiber (ADF), as well as the in vitro digestibility of dry matter (IVDMD) and in vitro digestibility of NDF (IVNDFD) and ADF (IVADFD), were determined according to [29].

2.6. Statistical Analysis

A completely randomized design with a 2 × 4 factorial arrangement was used. An analysis of variance was performed with the GLM procedure of SAS v9.3 [30]. The adjusted model included the effects of treatment and fermentation time, and the interaction between these two factors. The following equation was applied:
Yijk = μ + Ti + Hj + THij + εijk
where Yijk = the dependent variable, µ = overall mean, Ti = treatment effect (i = 1.2), Hj = effect of time (j =1, 2, 3, 4), THij = interaction effect, and εijk = experimental error code.

3. Results and Discussion

Table 2 shows the results of the in vitro ruminal fermentation of corn stover, with the addition of Levica 27 in the culture medium. There was no effect (p > 0.05) of the inclusion of the yeast when compared to the control treatment for pH, N-NH3, % NDF, %ADF, % IVDMD, % IVNDFD, and % IVADFD; but there was a difference (p < 0.05) in the incubation times for these variables.
The culture pH showed no interaction between the treatments and the incubation time (p > 0.05), nor an effect of the treatment (p > 0.05), but between the different incubation times (p < 0.05). Nevertheless, the pH remained at values that were close to neutrality throughout the experiment (6.7–6.9), which are considered to be within the physiological range of the ruminal fluid pH [31]. These values coincide with the ruminal pH that is recorded when low-quality fibrous substrates are supplied to the cattle, such as the one used in this study, and they coincide with the results reported by [16,32] when they studied the effect of different yeast species on the in vitro ruminal fermentation of fibrous substrates. The lack of difference observed in the pH results may be due to the fact that when high-fiber diets are used, one of the microbial populations that establishes in the rumen are methanogenic bacteria, which use hydrogen (H2) and carbon dioxide (CO2) for the formation of methane. The use of these gases by these bacteria, especially H2, contributes to the stabilization of the ruminal pH [33].
The effects of yeast cultures on ruminal pH reported in the literature are variable, depending mainly on the experimental conditions [34]. Among the main factors that affect these results are the composition of the diets evaluated [4,35,36,37] and the yeast species used [16]. Authors such as Chaucheyras-Durand and Fonty (2008) [38] showed that yeasts can mainly increase the ruminal pH or decrease its variability when diets that are high in concentrate are used, due to a decrease in lactate concentration, which is attributed to the interactions that are established between yeast cells and the bacteria that metabolize lactate [39]. Chaucheyras et al. (1996) [40] demonstrated that under in vitro conditions, Saccharomyces cerevisiae cells were able to outnumber Streptococcus bovis cells when they competed for the use of sugars, which resulted in a lower lactate production by S. bovis.
Regarding the levels of NH3-N (Table 2), the molar concentration varied over time (p < 0.05); however, no differences were observed (p > 0.05) with the addition of yeast. Values of between 7 and 119 mmol/mL were obtained, which are considered as being high when they are compared to other similar studies where this concentration ranges between 5 and 12 mmol/mL with fibrous diets [16,41]. This difference is due to the presence of urea in the culture medium used for the growth of Levica 27, which was added as an inoculum in the in vitro ruminal fermentation bottles. In accordance with these results, Vallejo-Hernández et al. (2018) [4] found no effect with the addition of S. cerevisiae on NH3-N levels during ruminal fermentation when using a totally mixed ration in studies with goats, sheep, and steers. However, the ammonia levels found in that study were also high (66 mmol/mL), because the diet used contained 40 g of urea per kg of DM. On the other hand, Diaz et al. (2017) [35] reported increases in ammonia concentration when adding a yeast hydrolysate to ruminal fluid using a 1:1 ratio of forage and concentrate as substrate. However, Anjum et al. (2018) [36] found that the addition of S. cerevisiae decreased the concentration of this compound during the ruminal fermentation of buffaloes.
It was expected that with the addition of the Levica 27 culture, the NDF and ADF percentages would decrease, and the IVDMD, IVNDFD, and IVADFD percentages would increase; however, there was no variation. According to these results, Ferriere (2017) [41] found no effect of two commercial yeasts (Ganadero PLUS® and LEVUCELL SC-10®) when studying the digestibility percentages of NDF and ADF (43.88% and 39.84%, respectively) for 72 h of in vitro fermentation with alfalfa hay as a substrate, also coinciding with what was reported by Suntara (2020) [42], who found no differences in the apparent digestibility of DM, NDF, and ADF when adding two yeast strains (Pichia kudriavzevii and Candida tropicalis) in the in vitro ruminal fermentation of rice straw silage. Using corn stubble as a substrate, Angeles et al. (1999) [43] and Crosby et al. (2011) [44] also did not find improvements in ruminal fermentation when adding commercial yeast strains (S. cerevisiae) in increasing doses from 0.015 to 7 g/day, and the digestibility results range over 55–64% DM, 57–61% NDF, and 25.28% ADF. On the other hand, some studies showed improvements in the DM and NDF digestibility indicators when fibrous diets were evaluated, fundamentally because they increased the number of cellulolytic bacteria and fungi and their fibrolytic activities [16,36,45,46,47,48].
The effect of yeast cultures on the digestibility of DM, ADF, and NDF has been inconsistent in both in vitro and in vivo studies in calves and dairy cows. These inconsistencies are related, among other factors, to the yeast species studied [17,49,50] and to the type of diet used [47]. Regarding how the type of diet influences results, Roa et al. (1997) [51] pointed out that the quality of the forage impacts the digestion of the fiber when the addition of yeast cultures is evaluated, suggesting that the best responses are obtained with better-quality forages. In contrast, Castillo et al. (2016) [17] found that the addition of a culture of Candida norvegensis increased gas production, which is closely related to the digestibility of the fibrous components, both when they used alfalfa hay as a substrate and when they used oat straw. In that study, the influence of the yeast species was also observed, since the culture of C. norvegensis increased gas production with respect to S. cerevisiae. In relation to what was found by the latter authors, Dawson and Tricarico (2002) [52] suggest that yeast preparations are less effective when animals are fed well-balanced diets that favor the stability of the gastrointestinal microbial population, and that it is likely that they would have promising results with diets high in forages. Dawson and Hopkins (1991) [53] pointed out that not all yeast strains are capable of stimulating ruminal bacteria, since, out of a total of 50 strains evaluated, only seven strains were capable of stimulating the growth of fiber-digesting bacteria.
Table 3 shows the behavior of the VFA concentration. There was an interaction (p < 0.05) of treatment with sampling time for the molar concentration of each VFA and the C2:C3 ratio. The inclusion of Levica 27, when compared to the control treatment, increased the molar concentrations for total VFAs, and acetic and propionic acids in ruminal fluid only at 12 h of fermentation (p < 0.05), and a decreased C2:C3 ratio (p < 0.05) was also seen.
The optimal proportion of VFAs in the rumen should have an acetate concentration of 65 to 70%, propionate of 20 to 25%, and butyrate of 10 to 15% [37], whose values are in agreement with the percentages obtained in the current study, where the VFA averages were 66.1, 20.5, and 13.2% for acetate, propionate, and butyrate, respectively.
The results found in the literature regarding the molar concentrations of total or individual VFAs are also inconsistent [37,47,54,55]. Similar results to those found in the present study were reported by Oeztuerk et al. (2005) [31] and Kowalik et al. (2012) [56].
Corresponding to our results, when Ruiz et al. (2016) [16] studied the effect of Candida norvegensis on the in vitro ruminal fermentation of oat straw, they reported that the molar concentration of acetic, propionic, and butyric acids increased with respect to the control only between 8 and 12 h. These results, where the inclusion of yeast cultures in fibrous diets increased the molar concentration of total VFAs and acetic and propionic acids between 8 and 12 h, may be due to the fact that these cultures tend to reduce the lag time for bacteria [45,57] which is known as the inactive phase. The shorter this phase lasts, the faster the commencement of the activity of the ruminal bacteria, increasing the rates of digestion, but not increasing the extension of digestion by these microorganisms [57]. In this sense, the yeasts have their greatest effects in the first hours of fermentation. Callaway and Martin (1997) [58], when studying the effect of a yeast on the disappearance of cellulose, found that the best results were in the first 24 h, and there was no effect after 48 h.
Contrary to what was found in our study, Marrero et al. (2006) [32] reported no effect on the molar concentration of total VFAs when they evaluated different yeast strains in the ruminal fermentation of cows that consumed fibrous diets. Similar to this last author, Moya et al. (2009) [34] also did not observe differences with the inclusion of a S. cerevisiae culture with regard to the molar concentration of VFAs, or with regard to the C2:C3 ratio in Holstein heifers consuming a transition diet. The decrease in the C2:C3 ratio of the yeast treatment with respect to the control found in the current study (p < 0.05) is explained by the higher molar concentration of propionate in the yeast treatment, which results in changes in the molar proportion of propionate. Similar results where higher concentrations of propionate were found were reported by Lila et al. (2004) [57] and Ruiz et al. (2016) [16]. Lila et al. (2004) [57] reported that one of the main effects of yeast cultures is to increase the concentration of propionate at the expense of the concentration of acetate. This could be due to peptide fractions in yeast cells that stimulate the growth of Megasphaera elsdenii, the main lactate-consuming microorganism in the rumen, which uses lactate to produce propionate through the acrylate pathway [37]. In addition, yeast cultures provide thiamin, glucans, mannoproteins, and organic acids, which stimulate the growth of microorganisms that digest fiber and consume lactic acid [31].
The results suggest that Levica 27 could improve the use of food energy for production purposes, due to the increase in the gluconeogenic potential of the diet from the higher proportion of propionate. It is well known that propionate is the only VFA that contributes to hepatic gluconeogenesis, in addition to being the most energy-efficient because its production is indirectly related to methanogens through the use of metabolic hydrogen [48]. The stimulating effect that yeast cultures have on the increased production of total and individual VFAs when fibrous diets are used can be explained by their own mechanisms of action, since it has been shown that live yeasts consume the oxygen that enters into the rumen through feed, rumination, or salivation, and this brings about better anaerobic conditions, which facilitate the growth of most anaerobic microorganisms, such as bacteria and cellulolytic fungi [58,59]. A decrease in the redox potential in lambs, sheep, and cattle was reported by Chaucheyras-Durand et al. (2012) [60] when they included live yeasts in the feeds of these animals.

4. Conclusions

The addition of Levica 27 for in vitro ruminal fermentation with a low-quality fibrous substrate had no effect on the ruminal pH or NH3-N concentration, nor on the indicators of the fiber degradability and digestibilities of DM, NDF, and ADF. However, after 12 h of fermentation, the total production of VFAs as well as the molar amounts of acetic and propionic acids were higher when the yeast was used.

Author Contributions

Conceptualization, Y.C.-C. and O.R.-B.; writing—original draft preparation, B.G.M.; writing—review and editing, Y.C.-C., O.R.-B. and F.C.R.; supervision, O.R.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this research are available within the article.

Acknowledgments

The authors gratefully acknowledge scientific support from Robin Anderson and Michael Hume.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Chemical composition of corn stover used as substrate for in vitro ruminal fermentation.
Table 1. Chemical composition of corn stover used as substrate for in vitro ruminal fermentation.
NutrientDry Matter (%)
Acid detergent fiber37.75
Ash8.35
Neutral detergent fiber67.2
Crude protein5.90
Crude fiber31.07
Dry matter91.44
Total sugars13.34
Table
Table 2. Effect of Pichia guilliermondii strain Levica 27 on the in vitro ruminal fermentation of corn stover.
Table 2. Effect of Pichia guilliermondii strain Levica 27 on the in vitro ruminal fermentation of corn stover.
Variable 1TreatmentSEp-Value
ControlLevica 27TreatTimeTreat × Time
pH
06.88 a6.91 a0.050.1<0.00010.68
66.70 b6.73 b
126.87 a6.92 a
246.94 a6.94 a
NH3-N (mmol/mL)
015.74 d7.81 d4.180.82<0.00010.91
630.50 c33.42 c
12107.46 b98.07 b
24113.97 a119.78 a
NDF 2 (%)
664.26 a63.18 a3.080.170.0050.68
1263.75 ab60.40 ab
2458.57 b57.66 b
ADF (%)
635.5934.882.050.710.120.85
1235.5734.98
2433.1433.50
IVDMD (%)
636.79 b37.88 b3.090.170.0050.68
1237.33 ab40.67 ab
2442.49 a43.40 a
IVNDFD (%)
626.41 b27.94 b2.730.240.020.93
1228.28 ab29.99 ab
2430.98 a31.76 a
IVADFD (%)
612.96 b15.05 b3.750.160.010.92
1215.69 ab18.66 ab
2419.73 a21.26 a
1 Means (n = 4) in columns with different letters for the time indicate significant differences at p < 0.05. 2 Neutral detergent fiber (NDF), acid detergent fiber (ADF), in vitro digestibility of dry matter (IVDMD), and in vitro digestibility of NDF (IVNDFD) and ADF (IVADFD).
Table
Table 3. Effect of Pichia guilliermondii strain Levica 27 during in vitro ruminal fermentation on the production of volatile fatty acids.
Table 3. Effect of Pichia guilliermondii strain Levica 27 during in vitro ruminal fermentation on the production of volatile fatty acids.
Volatile Fatty Acids1Treatment
Control Levica 27		SEp-Value
Acetic
0 h5.9 c5.90.500.03
6 h6.7 c7.6 c
12 h8.8 c12.2 b
24 h18.6 a18.0 a
Propionic
0 h1.1 d1.1 d0.07<0.0001
6 h2.3 c2.5 c
12 h2.1 c4.2 b
24 h6.1 a6.3 a
Butyric
0 h0.5 c0.5 c0.310.0008
6 h1.6 bc1.7 bc
12 h1.5 bc2.8 b
24 h10.9 a7.2 a
Total VFAs
0 h7.4 d7.4 d0.590.0005
6 h10.7 cd11.7 c
12 h12.5 c19.2 b
24 h35.5 a31.4 a
C2:C3
0 h5.3 a5.3 a0.220.01
6 h2.9 c3.0 c
12 h4.1 b2.9 c
24 h3.1 c2.9 c
1 Means abcd (mmol/mL, n = 4) for each volatile fatty acid (VFA) with different letters indicate significant differences at p < 0.05.
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González Mora, B.; Ruiz-Barrera, O.; Castillo Rangel, F.; Castillo-Castillo, Y. Effect of Live Yeasts (Pichia guilliermondii) on In Vitro Fermentation of Corn Stover as a Fibrous Substrate. Fermentation 2023, 9, 17. https://doi.org/10.3390/fermentation9010017

AMA Style

González Mora B, Ruiz-Barrera O, Castillo Rangel F, Castillo-Castillo Y. Effect of Live Yeasts (Pichia guilliermondii) on In Vitro Fermentation of Corn Stover as a Fibrous Substrate. Fermentation. 2023; 9(1):17. https://doi.org/10.3390/fermentation9010017

Chicago/Turabian Style

González Mora, Bexy, Oscar Ruiz-Barrera, Francisco Castillo Rangel, and Yamicela Castillo-Castillo. 2023. "Effect of Live Yeasts (Pichia guilliermondii) on In Vitro Fermentation of Corn Stover as a Fibrous Substrate" Fermentation 9, no. 1: 17. https://doi.org/10.3390/fermentation9010017

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