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Effect of Maillard reaction products on ruminal and fecal acid-resistant E. coli, total coliforms, VFA profiles, and pH in steers¹

Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-1600

Abstract

Six ruminally cannulated Angus-cross steers (362 kg) were used in a replicated 3 x 3 Latin square design to determine effects of supplementing Maillard reaction products (MRP) on acid-resistant E. coli and coliform populations. Steers were fed roughage-based diets supplemented (DM basis) with either 10% soybean meal (SBM), 10% nonenzymatically browned SBM (NESBM), or 10% SBM top-dressed with 45 g of a lysine-dextrose Maillard reaction product (LD-MRP). Equal weights of dextrose, lysine hydrochloride, and deionized water were refluxed to produce the LD-MRP. The NESBM was manufactured by treating SBM with invertase enzyme, followed by heating to induce nonenzymatic browning. Steers were allowed slightly less than ad libitum access to diets fed twice daily and were adapted to their respective treatments within 10 d. On d 11, ruminal and fecal samples were collected at 0, 2, 4, 6, 8, and 12 h after feeding from each of the steers and transported to the laboratory for microbial analysis. Ruminal samples and feces were analyzed for pH and VFA, and both ruminal fluid and feces were tested for acid-resistant E. coli and total coliforms by incubating samples in tryptic soy broth adjusted to pH 2, 4, and 7. Ruminal pH and total VFA concentrations did not differ among treatments. The molar proportion of ruminal acetate was higher (P < 0.05) for steers receiving NESBM than for steers receiving SBM and LD-MRP. At pH 4, steers that received NESBM had lower (P < 0.05) ruminal populations of E. coli and total coliforms than steers that received SBM. No differences were observed for ruminal E. coli and total coliforms at pH 2 and 7. Fecal pH was lower (P < 0.05) for steers fed NESBM than for steers fed SBM or LD-MRP. Molar proportions of fecal acetate were lower (P < 0.05) and proportions of butyrate and isovalerate were higher (P < 0.05) for steers fed NESBM compared with steers fed SBM. Fecal E. coli at pH 4 was lower (P < 0.05) for steers fed NESBM than for steers fed LD-MRP. Fecal E. coli and total coliforms at pH 2 and 7 did not differ among treatments. Dietary MRP had limited effectiveness at decreasing acid-resistant coliforms in the rumen and feces of cattle. Acid resistance in coliforms may depend on protein availability.

Key Words: Cattle • E. coli • Maillard Reaction Products

Introduction

Beef can become microbiologically contaminated when fecal material from cattle is transferred to the carcass during slaughter (Byrne et al., 2000). Enterohemorrhagic Escherichia coli (EHEC), including E. coli O157:H7, reside in the gastrointestinal tract of cattle (Whipp et al., 1994), and, when consumed by humans, EHEC can survive the human gastric barrier and proliferate in the lower gastrointestinal tract (Lin et al., 1996), causing illness and even death (Nataro and Kaper, 1998). Thus, identification of preslaughter interventions for reducing the number of acid-resistant fecal pathogens produced by cattle is warranted.

Heating of reducing sugars in the presence of free amino groups results in the formation of brown pigments called melanoidins, or Maillard reaction products (MRP). Production of MRP by nonenzymatic browning is commonly induced to effectively protect protein from ruminal proteolysis without reducing protein digestibility (Cleale et al., 1987). Maillard reaction products also act antimicrobially against certain bacterial species. The antimicrobial action of MRP is thought to result from its ability to sequester iron, which is essential for growth and survival of pathogenic bacteria, and to interfere with the bacterial uptake of serine, glucose, and oxygen (Einarsson et al., 1988). Marounek and Brezina (1993) observed that adding MRP to growth media could inhibit the growth of several species of ruminal bacteria. Stecchini et al. (1991) demonstrated that MRP inhibited the growth of food-poisoning bacteria, including Aeromonas hydrophila, Staphylococcus aureus, and Listeria monocytogenes. Additionally, Einarrson et al. (1983) and Einarrson (1987a) found that MRP prolonged the lag phase of E. coli. Our objective was to determine if sources of dietary MRP could effectively reduce the acid-resistant populations of E. coli and coliform in the rumen and feces of cattle.

Experimental Procedures

Procedures for this study were approved by the Kansas State University Institutional Animal Care and Use Committee. In the fall of 1999, six ruminally cannulated Angus-cross steers were used in a replicated 3 x 3 Latin square design. Steers were individually fed roughage-based diets and supplemented (DM basis) with either 10% soybean meal (SBM), 10% nonenzymatically browned SBM (NESBM), or 10% SBM top-dressed with 45 g of a lysine-dextrose Maillard reaction product (LD-MRP; Table 1). The NESBM and LD-MRP were manufactured according to Coetzer (2000). Briefly, NESBM was made by treating solvent-extracted SBM with an invertase enzyme to liberate reducing sugars, followed by heating to a core temperature of 105°C to promote browning. The LD-MRP was produced by mixing equal weights of dextrose, synthetic L-lysine, and deionized water and subsequently refluxing the solution under alkaline conditions for 4 h to induce nonenzymatic browning.

Approximately 100 mL of unstrained ruminal fluid and at least 10 g of feces from each steer were placed on ice and transported to the laboratory for bacterial enumeration. Sterile deionized water (20 mL; pH 6.7) was added to 10 g of feces to liquefy the fecal samples. Ruminal and fecal samples were homogenized for 2 min in a Stomacher Lab Blender 400 (Tekmar, Cincinnati, OH). To test for acid resistance, homogenized samples (1 mL) were added to filter-sterilized tryptic soy broth (9 mL) adjusted to pH 2, 4, or 7 with 85% lactic acid or 1 M NaOH. The pH 2 treatment was chosen because the human stomach is frequently at pH 2. The intermediate treatment (pH 4) was chosen because meat processors commonly use a pH 4 acid wash to decrease bacterial contamination on beef carcasses. After the acid treatments, ruminal and fecal samples were incubated for 1 h at 32°C. After 1 h, 1 M NaOH was added to the culture tubes to neutralize acidic samples exposed to pH 2 and pH 4. Serial dilutions were prepared in 0.1% sterile peptone diluent. Appropriate dilutions were plated in duplicate on E. coli Count Petrifilm (3M, St. Paul, MN) to enumerate E. coli and total coliform populations. All plates were incubated at 35°C for 24 ± 2 h and enumerated. Ruminal samples were analyzed in colony-forming units per milliliter. Fecal samples were dried at 105°C in a forced-air oven and the population counts recorded on the Petrifilm were adjusted to colony-forming units per gram of dry feces. Concentrations of VFA were measured using gas chromatography (Hewlett-Packard 5890A gas chromatograph, 183 x 0.635 cm column, Supelco SP 1200 packing, N₂ carrier at 80 mL/min, flame ionization detector at 225°C).

Ruminal and fecal E. coli, total coliform, pH, and VFA were analyzed as a split, split-plot design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). Effects of steer, period, and dietary treatment were in the whole plot, whereas sampling time and sampling time x dietary treatment were in the split plot. Acid treatment, acid treatment x sampling time, acid treatment x dietary treatment, and acid treatment x dietary treatment x sampling time were included in the split, split-plot design. The steer x period x dietary treatment interaction was the main-plot random variable and served as the error term. The steer x period x dietary treatment x sampling time interaction was used as the split-plot error term.

For statistical analyses, a value of half the detection limit was entered for samples in which a target population was not detected. The detection limit depended on whether the sample was of ruminal or fecal origin and the acid treatment applied to each sample.

Results and Discussion

The underlying hypothesis of this study was that dietary MRP may prove effective at decreasing the acid-resistant coliform in the ruminal and fecal contents of cattle. The results of ruminal pH, total VFA, and individual VFA proportions are presented in Table 2. Ruminal pH and total concentration of VFA were not different among treatments. The molar proportion of ruminal acetate was higher (P < 0.05) for steers fed NESBM than for steers fed the SBM and LD-MRP, but other VFA proportions were similar among treatments. A dietary treatment x time interaction (P < 0.05) occurred for total ruminal VFA (Figure 1). Steers fed NESBM had higher total VFA concentrations at 6 h after feeding compared with steers fed SBM or LD-MRP; however, this concentration dropped over time and at 8 and 12 h after feeding was lower for steers fed NESBM compared with steers fed SBM or LD-MRP. A dietary treatment x time interaction also occurred for ruminal pH (Figure 2). Steers fed LD-MRP had lower (P < 0.05) ruminal pH at 8 and 12 h after feeding compared with steers fed NESBM and pH was lower (P < 0.05) at 8 h compared with steers fed SBM. The lower ruminal pH at times further from feeding combined with the numerically higher VFA concentration suggests that microbial fermentation in the rumen persisted longer for steers receiving supplemental LD-MRP than for steers receiving supplemental NESBM or SBM. Einarsson (1987a) found that addition of MRP to the growth media extended the lag phase of E. coli, and Marounek and Brezina (1993) found that MRP lowered the growth rate and growth yield in most ruminal bacteria. Perhaps the LD-MRP altered the growth rate of ruminal microflora and caused a shift in fermentation and prolonged the breakdown of substrate in the rumen.

View larger version (14K): [in this window] [in a new window]   Figure 1. Concentrations of VFA in ruminal fluid collected from steers fed roughage-based diets supplemented with either 10% soybean meal (SBM ), 10% nonenzymatically browned SBM (NESBM ), or 10% SBM plus 45 g/(steer•d) of a lysine-dextrose Maillard reaction product (LD-MRP ). Six observations per treatment mean. Diet x time, P < 0.05, SEM = 5.5.

View larger version (14K): [in this window] [in a new window]   Figure 2. Ruminal fluid pH of steers fed roughage-based diets supplemented with either 10% soybean meal (SBM ), 10% nonenzymatically browned SBM (NESBM ), or 10% SBM plus 45 g/(steer•d) of a lysine-dextrose Maillard reaction product (LD-MRP ). Six observations per treatment mean. Diet x time, P < 0.05, SEM = 0.055.

The acid treatments used in this experiment altered the populations of E. coli and total coliform sampled from the rumen (shown later in Table 4). Essentially, pH 2 was completely inhibitory to all bacterial growth, regardless of dietary treatment. Escherichia coli and total coliform counts isolated from all steers averaged 2.3 log₁₀ cfu/mL and 3.0 log₁₀ cfu/mL, respectively, at pH 7. Brownlie and Grau (1967) and Diez-Gonzalez et al. (1998) report that cattle fed similar diets had ruminal E. coli populations of approximately 4 to 5 log₁₀ cfu/mL. Perhaps the different media or culturing techniques used by these researchers resulted in a better recovery of coliforms.

Fecal pH, total VFA, and individual VFA proportions are presented in Table 3. Fecal pH was lower (P < 0.05) for steers fed NESBM than for steers that received SBM or LD-MRP. This can be attributed to the numerically higher fecal concentration of VFA that was observed in cattle that received NESBM. No treatment differences over time were observed for total fecal VFA (Figure 3). A treatment x time interaction occurred for fecal pH (Figure 4). Steers receiving NESBM had lower (P < 0.05) fecal pH at 2, 6, and 8 h after feeding compared to steers receiving SBM and had lower (P < 0.05) fecal pH at 8 and 12 h after feeding compared with steers that received LD-MRP. Steers receiving SBM had lower (P < 0.05) fecal pH at 12 h after feeding compared with steers fed LD-MRP.

View larger version (13K): [in this window] [in a new window]   Figure 3. Concentrations of total VFA in feces of steers fed roughage-based diets supplemented with either 10% soybean meal (SBM ), 10% nonenzymatically browned SBM (NESBM ), or 10% SBM plus 45 g(steer•d) of a lysine-dextrose Maillard reaction product (LD-MRP ). Six observations per treatment mean. Diet x time, P = 0.10, SEM = 7.9.

View larger version (12K): [in this window] [in a new window]   Figure 4. Fecal pH from steers fed roughage-based diets supplemented with either 10% soybean meal (SBM ), 10% nonenzymatically browned SBM (NESBM ), or 10% SBM plus 45 g(steer•d) of a lysine-dextrose Maillard reaction product (LD-MRP ). Six observations per treatment mean. Diet x time, P < 0.01, SEM = 0.061.

Total populations of E. coli increased in number from the rumen to the feces by 2.5 to 3.7 log units across diets, when comparing samples incubated at pH 7. Total coliform count increased from the rumen to the feces by 2.9 to 3.2 log cycles at pH 7 (Table 4). Fecal E. coli and total coliform averaged across dietary treatments at pH 7 were 5.4 log₁₀ cfu/g and 6.0 log₁₀ cfu/g, respectively. This is similar to E. coli and coliform populations of 4.5 to 5.0 log₁₀ cfu/g of feces reported by Diez-Gonzalez et al. (1998) and E. coli populations of 6.4 to 6.7 log₁₀ cfu/g of feces reported by Jordan and McEwen (1998). The higher populations of coliforms in the feces compared to the rumen suggests that higher concentrations of VFA in the rumen (96 mM) vs. the feces (46 mM) inhibit E. coli and total coliform propagation. Previous research has demonstrated that animals offered ad libitum access to feed had higher VFA concentrations and subsequently lower E. coli populations compared to animals that were fasted (Grau et al., 1969; Rasmussen et al., 1993; Kudva et al., 1995).

Steers that were fed NESBM had lower (P < 0.05) E. coli populations at pH 4 compared with steers that received LD-MRP (Table 4). Steers that were fed LD-MRP had 1.0 log₁₀ cfu/g more E. coli at pH 4 than steers fed SBM or NESBM. No differences were observed based on dietary treatment for pH 2 and 7. Although the NESBM and LD-MRP both contain the brown melanoidin pigments, the products may inhibit bacteria differently. Einarsson (1987b) demonstrated that dissimilar MRP affect microbial populations differently as a result of their unique composition. The reducing sugars used in the manufacturing of NESBM were, most likely, glucose and fructose, and the reducing sugar used to make LD-MRP was exclusively glucose. Additionally, the amino groups that react with these reducing sugars could have been different. Soybean meal contains an array of AA. Einarrson (1987b) reported that MRP formed in mixtures containing arginine elicited a greater inhibitory effect than MRP found in mixtures containing histidine. Other researchers have demonstrated that some MRP are more effective at reducing gram-positive than gram-negative bacteria (Jemmali, 1969; Einarsson et al., 1983). Perhaps the LD-MRP was more selective for gram-positive bacteria and, by competitive inhibition, allowed the gram-negative E. coli and coliforms to proliferate.

Duncan et al. (2000) suggested that providing dietary protein supplements that increase protein flow to the ruminant small intestine may influence the survival of E. coli O157:H7 in the rumen. They implied that protein supplements may increase the availability of AA and permit E. coli O157:H7 to maintain its internal pH under the acidic conditions of the abomasum. By feeding the NESBM and LD-MRP we may have reduced the available AA supply in the rumen and subsequently reduced E. coli growth.

Similar to the ruminal samples, exposure to pH 2 eliminated nearly all E. coli and coliform populations. The acid treatments applied to these samples may have been too rigorous to depict differences due to dietary attributes. Yet, possibly by feeding a high-roughage diet, the steers in our study did not produce sufficient concentrations of VFA to cause ruminal and fecal E. coli and coliforms to develop tolerance to our highly acidic, pH 2 treatment. This would parallel the findings of Diez-Gonzalez et al. (1998) with cattle fed high-roughage diets.

Researchers have theorized that some strains of E. coli can become habituated to acid exposure, develop tolerance, and resist low-pH extremes that mimic the human gastric barrier (Gorden and Small, 1993; Benjamin and Datta, 1995; Diez-Gonzalez and Russell, 1999). Diez-Gonzalez et al. (1998) and Tkalcic et al. (2000) reported that cattle that were fed high-concentrate diets, but not high-roughage diets, supported the growth of acid-resistant E. coli at low-pH treatments. By feeding a high-concentrate diet, perhaps we could have developed greater populations of E. coli or coliform that would have been resistant to pH 2. However, previously we found that increasing ruminal and fecal acid concentrations and lowering pH by feeding high-concentrate diets to steers do not increase acid-resistant E. coli (Sindt et al., 2002).

The uniqueness of acid resistance and its relationship to commensal and pathogenic strains of E. coli has been debated. Some research has tried to demonstrate that EHEC is more acid-resistant than generic E. coli. However, Duffy et al. (2000) studied strains of generic E. coli and EHEC in an acid-resistance challenge and reported that generic E. coli could be used as a model for studies designed to test compliance or standards for controlling EHEC. Additionally, McClure and Hall (2000), in a review, state that "there are no definitive studies to support the commonly held belief that E. coli O157:H7 is any more acid tolerant than other E. coli present as part of the normal enteric flora." Even though we did not measure the acid resistance of E. coli O157:H7, we feel that evaluating the populations of acid-resistant generic E. coli and total coliform provides inclusive information that encompasses several pathogenic strains of E. coli and other enteric pathogens and is an appropriate model to determine strategies for controlling acid-resistant, pathogenic, enteric pathogens.

Implications

Dietary supplements containing Maillard reaction products had limited effects on the populations of acid-resistant E. coli and coliform populations in the ruminal fluid and feces of steers. The effectiveness of Maillard reaction products at decreasing acid-resistant coliform may depend on the composition of the product. Establishment of and resistance in coliform bacteria may be dependent on protein availability.

Footnotes

¹ Article No. 02-269-J from the Kansas Agric. Exp. Stn.

² Correspondence: Call Hall, Room 133 (phone: 785-532-1204; fax: 785-532-5681; e-mail: jdrouill{at}oznet.ksu.edu).

Received for publication January 14, 2003. Accepted for publication November 19, 2003.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sindt, J. J. Articles by LaBrune, H. J. Search for Related Content PubMed PubMed Citation Articles by Sindt, J. J. Articles by LaBrune, H. J.