The objective of this study was to examine the effects of Aspergillus oryzue fermentation extract (Amaferm) on the in vitro ruminal fermentation of coastal bermudagrass, soluble starch and amino acids. Mixed ruminal microorganisms were incubated in anaerobic media for either 24 h (Amaferm alone, soluble starch, amino acids) or 48 h (bermudagrass). Amaferm was added to the incubation bottles (n = 4) at concentrations of 0, .4 or 1.0 g/liter. When mixed ruminal microorganisms were incubated with only Amaferm, the 1.0 ghter concentration increased the production of hydrogen (€32; P c .001), methane P < .Ol), acetate (P c .05). butyrate (P c .Ol), total VFA (P c .05) and NH3 (P c .05). Addition of both levels of Amaferm to soluble-starch (P c .15), acetate (P fermentations tended to enhance the production of H2 (P < .ll), c .29) and total VFA (P < .19); propionate production was increased (P c .lo) by 1.0 g/ liter Amaferm, resulting in a decrease (P c .05) in the acetate:propionate ratio. Fermentation of amino acids plus 1.0 ghter Amaferm enhanced the production of acetate (P c .OS), propionate (P< .05), valerate (P c .01) and total VFA (P< .lo) and decreased the acetate:propionate ratio (P < .05). In addition, NH3 production tended (P < .19) to increase with both levels of Amaferm. When bermudagrass was the substrate, few changes in fermentation products were observed with Amaferm treatment. However, 1.0 D t e r Amaferm decreased the digestibility of NDF (P c .05) and ADF (P < .12) by 8.1 and 10.4%,respectively. Amaferm stimulated the in vitro mixed ruminal microorganism fermentation of starch, bermudagrass and amino acids to different degrees. High levels of Amaferm had a detrimental influence on the digestion of bermudagrass by mixed ruminal microorganisms. (Key Words: Rumen Microorganisms, Feed. ,Additive, Fiber, In Vitro Culture, Fermentation.)


J. Anim Sci. Introduction

In the 1960s, several studies examined the effects of dietary enzyme supplements on

‘Reference to a company or a product does not imply approval or recommendation of the product by the Univ.of Georgia to the exclusion of other products that also may be suitable. 2Supported by BioZyme Enterprises Inc., St. Joseph, MO and the Univ.of Georgia Agric. Ekp. Sta. %ept. of Anim and Dairy Sci. 4Dept. of Microbial. Received August 7, 1989. Accepted November 3.1989.


ruminant feedstuff utilization and performance (Burroughs et al., 1960; Leatherwood et al., 1960; Theurer et al., 1963; Rust et al., 1965). The results were inconsistent. Burroughs et al. (1960) reported that weight gains were increased 7% in beef cattle under feedlot conditions, but other researchers observed no changes in weight gain or feed digestibility (Leatherwood et al., 1960; Theurer et al., 1963). Consumer concern regarding use of antibiotics and other growth stimulants in production livestock has grown in recent years. Therefore, interest by livestock producers has increased in evaluating the effects of probiotics on rumi-


Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018




nant performance. Fuller (1989) defines a probiotic as "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance." Nonbacterial probiotics added to ruminant diets generally consist of Succharomyces cerevisiue and(ur) Aspergillus oryzae cultures. Limited research has shown that probiotics increase plant cell wall and CP digestion and alter pH and VFA concentrations in the rumen (Arambel et al., 1987; Wiedmeier et al., 1987; Judkins and Stobart, 1988; Gomez-Alarcon, 1988; Harrison et al., 1988). No research, to our knowledge, has examined the effects of A. oryzue supplements on fermentation of bermudagrass or pure substrates by mixed ruminal microorganisms. Therefore, experiments were designed to evaluate the effects of an A. oryzue fermentation extract on the in vitro ruminal fermentation of coastal bermudagrass, amino acids and soluble starch. Materials and Methods

Ruminal contents were obtained from a 500-kg ruminally fistulated Angus steer fed 5.0 kg bermudagrass and 5.0 kg concentrate supplement once daily. The ruminal contents were obtained 1.5 h after feeding and squeezed through eight layers of cheesecloth into an Erlenmeyer flask with an @-free headspace. The flask was not disturbed for 20 min (39'C), permitting feed particles to rise to the top of the flask Particle-free fluid from the flask was anaerobically transferred (20% vol/vol) to a medium (pH 6.7) containing 292 mg K2HP04, 240 mg KE?2PO4,480 mg (NH4)2sO4,480 mg NaC1, 100 mg MgS047H20, 64 mg CaC12.2H20, 4,000 mg Na2C03 and 600 mg cysteine hydrochloride per liter (Russell and Martin, 1984; Russell and Strobel, 1988; Martin et al., 1989). Particle-free fluid and medium were mixed and 40 ml was transferred anaerobically to 160-ml serum bottles that contained either .4 g soluble starch, Tryp ticases or coastal bermudagrass. h o t h e r set of serum bottles contained a combination of .2 g soluble starch plus .2 g Trypticase or .4 g



coastal bermudagrass plus .2 g Trypticase. The bermudagrass (1 mm particle size) was analyzed as having 65.6% NDF, 31.2% ADF, 3.3% lignin ( b r i n g and Van Soest, 1970) and 9.9% CP (AOAC, 1984). Weighed amounts of A m a f m 6 (A. oryzue fermentation extract) were added to achieve final concentrations of either .4 g/liter or 1.0 @iter. Current recommended usage levels for Amaferm in production ruminant diets are between 1 and 3' g per head daily. The bottles.were sealed with butyl rubber stoppers and aluminum caps to contain the gas pressure and placed in a 39'C water bath. After 24 h (Amaferm alone, soluble starch, Trypticase) or 48 h (bemudagrass) of incubation, .5 ml of gas was removed from each of the bottles, and hydrogen and methane were measured on a Gow Mac thermal conductivity series 580 gas chromatograph7 equipped with a Porapak Q column7 (60'C, 20 ml/mjn N2 carrier gas). The bottles then were uncapped, and the pH was measured immediately with a pH meter. Bottles then were emptied into centrifuge tubes and centrifuged (l0,OOO x g, 4'C, 15 min) and the cell-free supernatant fluids were stored at -2o'C. Fiber components were analyzed by methods described previously (Goering and Van Soest, 1970). Volatile fatty acids in supernatant fluid samples were measured by GLC using a Varian model 3400 gas chromatograph* (colu m n temperature = 120'C, detector temperature = 175.0 equipped with an autosampler and a GP 10% SP-1200/1% H$O4 80/100 mesh size Chromosorb W AW columng (Anonymous, 1975). Ammonia was measured by a modified colorimelric method (Chaney and Marbach, 1962; Russell and Jeraci, 1984). AU experiments were performed on duplicate days with two experiments per day (n = 4). Data were analyzed by a least squares means ANOVA using a GLM procedure (SAS, 1985) for a completely randomized design with two levels of Amaferm added. The statistical model contained effects due to level of Amaferm addition.

Resultsand Dlscusslon

%BL Microbiology Systems,Cockeysville. MD. 6siozyme Enterprises, hc., St. J O S ~ PMO. ~ 7Gow Mac Instrument Co., Bridgewater, NJ. *varianhtnunent ~ r o u ppalo , m o , CA. 9Supelc0, Jnc., Bellafonte, PA.

Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018

In the absence of exogenous carbohydrates or amino acids, Amaferm had little effect (P > .lo) on pH, isobutyrate, isovalerate, valerate or the acetate:propionate ratio (Table 1). Hydro-



gen (P < .001) and propionate (P < .22) production were increased slightly by both concentrations of Amaferm, and 1.0 O t e r (P< .Ol), acetate (P Amaferm stimulated c .05), butyrate (P < .Ol), total VFA (P < .05) and N H 3 production (P < .05). Similar results were observed upon incubation of a yeast culture with mixed ruminal microbes in the absence of added carbon and energy sources in vitro (Martin et al., 1989). To determine the effects of Amaferm on starch fermentation, mixed ruminal microorganisms were incubated with an excess (.4 g/ 40 ml media) of soluble starch (Table 2). As expected, the concentrations of most fermentation products were much higher than the concentrations observed in the absence of carbohydrates (Table 1). Amaferm increased propionate production (P < .lo) at the 1.0 g/ liter level; both levels decreased the acetate: propionate ratio (P < .OS). There was no change (P > .lo) in the other fermentation products with added Amaferm; however, the production of H2, q, acetate, butyrate and total ITA tended to increase with increasing concentrations of Amaferm. No change (P > .lo) in final pH was observed. Fnunholtz et al. (1989) observed that Amaferm treatment (.25 mg/ml) increased butyrate production by mixed ruminal microbes in vitro. Amafenn supplementation did not affect ruminal pH and VFA production in cows fed a 50% concentrate diet (Wiedmeier et al., 1987) or in wethers consuming a 10 or 25% grain diet (Judkins and Stobart, 1988). Equivalent concentrations of a yeast culture stimulated the in vitro mixed ruminal microorganism fermentation of soluble starch to a greater extent than Amaferm under similar experhental conditions (Martin et al., 1989). Adding Trypticase, an enzymatic hydrolyzate of casein that contains peptides (Russell et al., 1983; Chen et al., 1987) as the fermentation substrate increased concentrations of butyrate, isobutyrate, isovalerate, valerate and N H 3 (Table 3 vs fermentations without added amino acids Tables 1 and 2). Because Trypticase is approximately 18% branchedchain amino acids (compositional data from BBL Microbiology Systems5), the increase in branchedchain VFA and a substantial amount of the N H 3 production can be attributed to the fermentation of branchedchain amino acids by the mixed ruminal microbes (Russell and Jeraci, 1984; Russell and Martin, 1984).

Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018



I 7

I d




Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018




Amaferm at 1.0 @iter increased acetate (P < .05), propionate (P c .OS), valerate (P < .01) and total VFA (P < .lo), whereas the acetate: propionate ratio was reduced (P e .05). There were no changes (P > .lo) in the remaining fermentation products or in final pH. Although the differences were not significant, both .4 and 1.0 g/liter Amaferm increased NH3 production by more than 20%. Amaferm treatment (1.0 g/liter) also increased N H 3 production by 35% in the absence of exogenous amino acids (P < .05; Table 1). Frumholtz et al. (1989) observed that Amaferm increased N H 3 concentrations over 30% in vitro, suggesting that Amaferm increased proteolysis in vitro either by providing additional nutrients to the ruminal microorganisms or possibly via endogenous proteolytic activity of A. oryzue (Boing, 1983), as proposed by Arambel et al. (1987). Yeast culture also has been observed to increase N H 3 concentrations in vitro (Arambel et al., 1987; Martin et al., 1989). In vivo studies have shown that N H f l concentrations were not altered by Amaferm supplementation (Wiedmeier et al., 1987; Gomez-Alarcon, 1988; Judkins and Stobart, 1988), but Harrison et al. (1988) demonstrated that yeast culture decreased ruminal N H 3 concentrations in Holstein cows. The effects of Amaferm on the mixed ruminal microorganism fermentation of coastal bermudagrass was evaluated (Table 4). Both concentrations of Amaferm caused pH to decrease (P c .001); the 1.0 sfliter treatment increased CT& production (P < .05). Little change (P > .lo) in the other fermentation products was observed. Neutral detergent fiber (P c .OS) and ADF (P < .12) digestion were decreased 8.1 and 10.4%,respectively, by 1.0 @iter Amaferm, but the .4 s/llter level did not affect (P > .lo) the digestibility of these fiber components. Harrison et al. (1988) reported that yeast culture decreased cellulose disappearance by ruminal fluid in vitro. Other studies have shown increased fiber digestion by fungal treatments (Wiedmeier et al., 1987; Judkins and Stobart, 1988; Gomez-Alarcon, 1988), but some researchers have found no effect on fiber digestibility (Arambel et al., 1987; Frumholtz et al., 1989; Martin et al., 1989). When mixed ruminal microorganisms were incubated with soluble starch plus Trypticase (Table 5), production of butyrate, valerate, branchedchain VFA and total VFA was

Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018

greater than with soluble starch alone (Table 2). Generally, addition of .4 O t e r Amaferm stimulated the production of acetate (P c .05), propionate (P < .001), isobutyrate (P e .05), valerate (P < .01) and total VFA (P < .Ol) more than the 1.0 &ter level. Only H2 production was greater (P c .07) at the 1.0 g/ liter concentration than the .4 ghter concentration of Amaferm. Addition of Amaferm did not affect (P > .lo) NH3 production or final pH, but it tended to decrease the acetate:. propionate ratio (P c .15) and increase CT& (P < .15), butyrate (Pc .19) and isovalerate (P c 20).

Because cellulolytic ruminal bacteria require branchedchain VFA and valerate for growth (Hungate, 1966; Stewart and Bryant, 1988), and because concentrations of these VFA were low when bermudagrass was the substrate (Table 4), Trypticase and bermudagrass together were incubated with mixed ruminal microorganisms (Table 6). Incorporation of Trypticase into the incubation medium with bermudagrass resulted in a greater than fourfold increase in the concentrations of isobutyrate, isovalerate and valerate compared with incubations that contained only bermudagrass (Table 4). In addition, N H 3 concentrations were increased over sevenfold. h e vious research has shown that N H 3 concentrations between 50 and 235 mg/liter were sufficient for microbial protein synthesis (Satter and Slyter, 1974; Mehrez et al., 1977; Russell and Strobel, 1987), so N H 3 probably was not limiting in our bermudagrass incubations (Table 4). Even though branched-chain VFA and valerate production were enhanced by adding Trypticase to bermudagrass (Table 6), the extent of NDF and ADF digestion remained unchanged (Table 4 vs Table 6). Amaferm treatment did not alter the fermentation products (P > .lo); however, 1.0 g/liter Amaferm decreased digestion of NDF (P < .lo) and ADF (P < .05)by 6.1 and 5.4%, respectively (Table 6). As observed with only bermudagrass (Table 4), the .4 @ter level did not influence (P > .lo) digestion of NDF and ADF. Therefore, increasing concentrations of valerate and branchedchain VFA by adding Trypticase did not overcome the decrease in. digestibility of NDF and ADF in Amafermtreated incubations. These results suggest that high levels of Amaferm exert an inhibitory effect on bermudagrass digestion by mixed



Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018



ruminal microorganisms. Furthermore, because the fermentation products were not decreased by 1.0 o t e r Amaferm (Tables 4 and 6), this compound appears to be providing unidentified growth factors for the ruminal microbes. Data supporting this hypothesis are shown in Table 1, where it can be seen that production of many of the fermentation products was stimulated by 1.0 @liter Amaferm in the absence of added carbohydrates or amino acids. In our experiments, production tended to be stimulated by Amaferm treatment (Tables l , 2, 4 and 5). These results contradict the in vitro results of Frumholtz et al. (1989), which showed a reduction in CH4 upon Amaferm treatment when mixed ruminal microorganisms were incubated using the rumen simulation technique (Rusitec). The reason for this difference is unknown, but may be explained in part by differences in culture conditions and(or) substrates that were used. In addition, Frumholtz et al. (1989) found that ruminal protozoal numbers were reduced 45% by Amaferm treatment. Because methanogenic bacteria have been found to be associated with ruminal protozoa (Krumholz et al., 1983; Stumm and Zwart,1986; Veira, 1986), some of the decrease in CH4 may be due to a decrease in protozoal numbers. Enumeration of protozoa was not done in our studies. Implications Amaferm stimulated in vitro mixed ruminal microorganism fermentation of amino acids, starch and bermudagrass to varying degrees. Generally, Amaferm treatment tended to increase the production of most fermentation products. These results suggest that Amaferm is providing additional unidentified growth factors to the ruminal microorganisms. Even though the in vitro mixed ruminal fermentation was enhanced by Amaferm, little change in final pH was observed except for a small decrease in the bermudagrass incubations.. Incorporation of 1.0 ghter Amaferm into bermudagrass incubations decreased digestion of NDF and ADF. Therefore, high levels of Amaferm had a detrimental influence on the digestion of bermudagrass by a mixed ruminal microbial population. Literature Cited

At~ouymous.1975.GC separation of VFA C2425. Bull. 7498. Supelco, Inc., Bellefonte, PA. AOAC. 1984. Official Methods of Analysis (14th Ed.).

Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018



on feed utilizationby ruminants.J. Dairy Sci. 43:1460. AssociationofOfficial Analyticalchemists, WashingMartin. S.A., D. J. Nisbet and R G. Dean. 1989.Influence ton, DC. of a commercial yeast supplement on the in vitro Arambel, M. J., R D. Wiedmein and J. L. Walters. 1987. ruminal fermentation. Nu@. Rep. Int. 40:395. Influence of donor animal adaptation to added yeast culture and/or Aspergilfusoryzuc fermentation extract Mehrez, A. Z., E.R 0rskov and I. McDonald. 1977.Rates of rumen famentation in relation to ammonia COIICenon in vitro rumen fermentation. Nut. Rep. Int. 35:433. tration. Br. J. Nu@. 38:437. B o a , J.T.P.1983. Enyme production. In: G. Reed @d.) Industrial Microbiology (4th Ed.). pp 685-689.AVI Russell, J. B. and J. L. J a a c i 1984. Effect of carbon monoxide on fermentation of f i k , starch, and amino PuMishing Co., Westport, CT. acids by mixed rumen miaoorganisms in Vitro. Appl. B m u g h s , W., W. Woods.S. A. Ewing. J. Grdg and B. Jinviron. Microbiol. 48:211. Theurcr. 1960. Enyme additions to fattening cattle Russell, J. B. and S. A. Martin. 1984. Effects of various dons. J. Anim. Sci. 19458. methane inhibiton on the fermentation of amino acids Chaney. A. L.and E. P.Marbach 1962.Modified reagents by mixed m e n microorganismsin vitro. J. Anim.Sci. for determination of urea and ammonia.Clin. Chem. 8: 591329. 130. Chen, G..H. J. Strobel,J. B. Russell and C. J. Sniffen. 1987. Russell,J. B., C. J. Sniffen and P.J. Van Socst. 1983.Effect of carbohydrate limitationon degradation and utilizaEffect of hydrophobicity on utilization of peptides by tion of casein by mixed rumen bacteria. J. Dairy Sci. ruminalbacteria in vitro. Appl. Environ. Microbiol. 53: 66:763. 2021. Frumholk, P. P.,C. J. Newbold and R J. Wallace. 1989. Russell, J. B. and H. J. Strobel. 1987. Concentration of ammonia m s s cell membranes of mixed rumen Irdluence of Aspergillus oryzac fermentation extract bacteria. J. Dairy Sci. 70970. on the fmentation of a basal ration in the rumen simulation technique (Rusitec). J. Agric. Sci. (Camb.) Russell, J. B. and H. J. Strobel. 1988.Effects of additiveson m vitro Nminal fermentation: A comparison of 113:169. monmsinand bacitracin, anotherpunpositive antibiMer, R. 1989.Probiotics in man and animals. J. Appl. otic. J. Anim. Sci 66552. Bacteriol. 66:365. Goering. H. K. and P. J. Van Saesl. 1970.Forage fiber Rust, J. W., N. L. Jacobson, A. D. McGilliatd and D. K. Hotchkiss. 1965. Supplementation of dairy calf diets analysis (apparatus, reagents, procedures, and some with enzymes. II. Effect on nutrient utilizationand on applications). Agric. Handbook 379. ARS, USDA, composition of rumen fluid. J. Anim. Sci. 24:156. washington, Dc. Gomez-Alarcon,RAG. 1988.Eff- of Aspergillus oryzac SAS. 1985.SAS User’s Guide: Statistics. SAS Inst., Inc., on Milk Production, Feed Utilizaton and Rumen cary, NC. F~~mentation inLectawDairy COWS.Ph.D. Dbserta- S a m , L. D. and L. L. Slyter. 1974.Effect of ammonia concentration on m e n microbid protein production tion. Univ. of Arhnn, Tucson. in vitro. Br. J. Nu@. 32:199. Harrison, G. A., R. W. Hemken, K. A. Dawson, R. J. Harmon andK. B. Barker. 1988.Influence of addition Stewart, C. S. and M. P. Bryant. 1988.The rumen bacteria. In: P. N. Hobson (Ed.) The Rumcn Microbial of yeast culture supplements to diets of lactating cows Ecosystem. pp 21-75. Elsevier Science Publishing on ruminal famentation and microbial populations. J. Co., Inc., New Yo&. Dairy Sci. 719967. Hungate, R. E. 1966. The Rumen and Its Microbes. pp Stamm,C. K. and K. B. Zwart 1986.Symbiosisof protozoa with hydrogemutilizing methanogem. Microbiol. Sci. 36-54. Academic Press, New York. 3:lOO. Judkins. M.B. and R H. Stobart. 1988.Influence of two levels d enzymepreparation on ruminal fermentation, Theurer, B., W.Woods and W. Burroughs. 1963.Influence of enzyme supplements in lamb fattening rations. J. particulate and fluid passage and cell wall digestion in Anim. Sci. 22150. wether lambs consuming either a 10% or 25% grain Veira.D.M. 1986.Theroleof~attprotozoainnutritionof diet. J. Anim. Sci. 66:lOlO. the ruminant. J. Anim. Sci. 63:1547. Krumholz, L.R.. C. W. Forsbcrg and D. M. Veira. 1983. Association of methanogenic bacteria with rumen Wiedmeier, R.D., M.J. Arambel and J. L. Walten. 1987. Effect of yeast culm and AspergUfus oryzue famenprotozoa. Can. J. Microbiol. 29:676. tation extract on ruminal characteristics and nutrient Ltathawood, J. M., R D. Mochrie and W. E. Thomas.1W. digestibility. J. Dairy Sci. 702063. Some effects of a supplemamy cellulase preparation

Downloaded from https://academic.oup.com/jas/article-abstract/68/7/2142/4704875 by Univ of California Library - Bioscience & Natural Res Library user on 05 August 2018

Effects of Aspergillus oryzae fermentation extract on fermentation of amino acids, bermudagrass and starch by mixed ruminal microorganisms in vitro.

The objective of this study was to examine the effects of Aspergillus oryzae fermentation extract (Amaferm) on the in vitro ruminal fermentation of co...
595KB Sizes 0 Downloads 0 Views