Agricultural and Food Science, Vol. 14 (2005): 311–324 311 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. © Agricultural and Food Science Manuscript received March 2005 Effects of some organic acids and salts on microbial fermentation in the digestive tract of piglets estimated using an in vitro gas production technique Kirsi Partanen MTT Agrifood Research Finland, Animal Production Research, Swine Research, Tervamäentie 179, FI-05840 Hyvinkää, Finland, e-mail: kirsi.partanen@mtt.fi Taina Jalava MTT Agrifood Research Finland, Animal Production Research, Animal Nutrition, FI-31600 Jokioinen, Finland An in vitro gas production technique was used to screen different organic acids (formic, propionic, lactic, citric, and fumaric acid), organic salts (calcium formate, potassium sorbate, and sodium benzoate), and in- organic phosphoric acid for their ability to modulate microbial fermentation in the digestive tract of piglets. For the incubation, 40 ml of culture medium (53% buffer, 45% frozen ileal digesta, and 2% fresh faeces) was dispensed in vessels containing 5 ml of buffer, 0.5 g of feed, and 20 µl of liquid or 20 mg of solid acidifiers. Gas production was measured every 15 min during the 24 h incubation at 39°C, and a Gompertz bacterial growth model was applied to the gas production data. Formic acid was the only acid that reduced the maximum rate of gas production (µm) compared to that in the control treatment (P < 0.05). The µm was slower in vessels with formic acid than in those with calcium formate, citric acid, and potassium sorbate (P < 0.05). Calcium formate increased the µm compared to the control treatment (P < 0.05). The maximum volume of gas produced and the lag time did not differ between different acidifiers (P > 0.05). When inves- tigating formic-acid-based mixtures that contained 1–5% of potassium sorbate and/or sodium benzoate, the estimated parameters for the Gompertz growth model did not differ from those for treatments with plain formic acid (P > 0.05). However, concentrations of total volatile fatty acids, acetic acid, propionic acid, and n-butyric acid were reduced by all the mixtures (P < 0.05), but not by plain formic acid (P > 0.05). In con- clusion, organic acids and salts were found to differ in their ability to modulate microbial fermentation in the digestive tract of piglets. Mixing formic acid with potassium sorbate or sodium benzoate changed fer- mentation patterns, and the possibility to use them to enhance the antimicrobial effect of formic acid should be investigated further in vivo. Key words: pigs, fermentation, organic acids, organic salts 312 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation Introduction In recent years, interest in the use of organic acids and their salts as alternatives to antibiotic growth promoters has increased dramatically. The addition of organic acids to swine diets has been shown to enhance growth and the feed to gain ratio of weaned piglets and fattening pigs (Kirchgessner and Roth 1988, Partanen and Mroz 1999). Although the exact mechanisms of the growth-promoting effect of or- ganic acids are not known, it seems that their growth- promoting effects are primarily due to their effects on gastrointestinal microflora. Organic acids appear to modulate microbial activity and fermentation in the gastrointestinal tract of pigs (Jensen 1998). Changes in microbial counts (Maribo et al. 2000a, b, Canibe et al. 2001) and microbial metabolite con- centrations (ammonia, lactic acid, and volatile fatty acids) have been found primarily in the stomach and small intestinal contents of pigs fed diets sup- plemented with organic acids (Roth et al. 1992, Canibe et al. 2001, Partanen et al. 2001a). The changes in microbial fermentation could result in nutrients being diverted from microbes to the animal (Jensen 1998), as well as improved apparent digest- ibility of nutrients, particularly that of protein and fat (Partanen et al. 2001a). Because of the high costs of animal experiments, an in vitro method could be a useful tool to screen different organic acids for their effects on microbial fermentation in the diges- tive tract of pig. The cumulative gas production technique has been successfully used to evaluate the effects of organic acids on caecal fermentation in pigs (Piva et al. 1999, 2002). Our aim was to use the in vitro gas production technique to study to what extent different organic acids, organic salts, and acid mixtures can modulate microbial fermentation in the digestive tract of piglets. The fermentation me- dium contained ileal digesta and feed as the substrate and faeces from young piglets as the inoculum. Material and methods The experimental protocol was reviewed and approved by the MTT Animal Care and Use Committee. Animals, diets, and the collection of digesta and faeces Ileal digesta was collected from a cannulated, growing pig (Finnish Landrace) when its body weight was between 50 and 80 kg. When its body weight had been 39 kg, this pig had been fitted with a PVC cannula at the caecum according to the post-valve T-cannulae technique (van Leeu- wen et al. 1991). Premedication and anaesthesia were carried out, and the pig was housed as de- scribed by Partanen et al. (2001b). The pig had two weeks time to recover from the surgery before it was fed a grower diet based on barley, wheat bran, and soya bean meal (Table 1). The pig was fed twice a day (0600 and 1800) and it was given 100 g of feed per kg of metabolic body weight (BW0.75) per day. Water was available ad libitum. Ileal digesta was collected 3–4 h after the morning meal by attaching a plastic bag with a rubber band to the barrel of the opened cannulae. The bag was removed immediately when a digesta pulse en- tered the bag. The bag was emptied under constant CO2 flow into a bottle that was frozen within 30 min after the initiation of the digesta collection. Because freezing stops microbial activity in di- gesta, faeces from piglets were used as inoculum. Faeces were collected from 2–4-week-old suck- ling piglets from six litters (Finnish Landrace) that had free access to creep feed containing no antimicrobials (Table 1). Faeces were collected in the morning and transported to the laboratory in anaerobic bags (BBL® GasPak Pouch™ System, Becton Dickinson Microbiology Systems, USA) within 2 h of collection. In vitro fermentation We used an automatic batch fermentation system that consisted of a thermostatically controlled wa- ter bath, magnetic stirrers (Variomag Telesystem HP 15 P, H + P Labortechnik AG, Munich, Ger- many), solenoid valves (121M13 fitted with coil 488980), pressure transducers (142PC05D, Hon- eywell, Inc., Minneapolis, USA), and a modular 313 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. Table 1. Ingredients and calculated composition of diets fed to pigs from which ileal digesta and faecal inoculum were collected for in vitro fermentation. Diet of pig from which ileal digesta was collected Diet of piglets from which faecal inoculum was collected Ingredients, g per kg of feed Barley 653.3 378.6 Wheat 400.0 Wheat bran 150.0 – Oat meal – – Soya bean meal 153.5 106.1 Low lactose whey powder (lactose 380 g kg-1) – 30.0 Fish meal – 60.0 Rapeseed oil 18.9 – Limestone 8.1 9.7 Monocalcium phosphate 2.6 1.6 L-Lysine HCl 0.6 1.7 Mineral and vitamin premix1 13.0 12.4 Calculated composition Net energy, MJ kg-1 9.1 9.2 Crude protein, g kg-1 160 186 Apparent ileal digestible lysine, g kg-1 6.9 9.3 Apparent ileal digestible threonine, g kg-1 4.2 5.6 Apparent ileal digestible methionine+cystine, g kg-1 4.2 5.6 Calcium, g kg-1 6.9 9.0 Phosphorus, g kg-1 6.2 5.5 1 Ten grams of premix contained the following minerals and vitamins: 1.8 g Ca, 0.6 g P, 0.4 g Mg, 2.5 g NaCl, 79 mg Fe, 17 mg Cu, 70 mg Zn, 18 mg Mn, 0.21 mg Se, 0.17 mg I, 3980 IU vitamin A, 398 IU vitamin D3, 38 mg vitamin E, 1.5 mg thiamin, 3.6 mg riboflavin, 2.1 mg pyridoxine, 15 µg vitamin B12, 0.15 mg biotin, 11 mg pantothenic acid, 15 mg niacin, 1.5 mg folic acid, and 1.5 mg vitamin K. programmable logic controller (MELSEC AnS, made by Mitsubishi Electric Europe GmbH, In- dustrial Automation, Ratingen, Germany). The AnS unit was controlled by the FactoryLink pro- gram (Beijers, G & L Beijer Electronics AB; Malmö, Sweden). The system was protected against electric power problems by Powerware Prestige 6000 uninterruptible power supply (Exide Electronics Oy, Espoo, Finland). The fermentation buffer (pH 7.78) was pre- pared according to Breves et al. (1991): 25 mmol NaHCO3, 1.0 mmol NaH2PO4·H2O, 2.0 mmol Na2SO4, 115 mmol NaCl, 10 mmol KCl, 2.5 mmol MgCl2, 2.5 mmol CaCl2, and 5.0 mmol NH4Cl per litre of distilled water. The buffer was warmed to 39°C and saturated with CO2 for 2 h before use. Frozen ileal digesta was thawed and diluted with fermentation buffer 1:2 using a Waring Laboratory Blender (GWB, USA) for 60 sec. Fresh piglet fae- ces used as inoculum were diluted with buffer 1:5 and mixed with the Waring Laboratory Blender for 60 sec. Faeces-buffer and digesta-buffer mixtures were mixed 1:10 so that the final mixture contained 2% faeces, 45% ileal digesta, and 53% buffer. The mixing ratio of digesta and buffer was adopted from the study of Piva et al. (2002). The concen- tration of faecal inoculum in fermentation fluid was modified based on the study of Knarreborg et al. (2002). The culture medium was sampled to de- termine the lactic acid, volatile fatty acids (VFA), and ammonia concentrations at time zero. For the incubation, 40 ml of culture medium was dispensed into a 100-ml vessel that contained a magnetic bar, 0.5 g of basal diet (ground to pass through a 1-mm sieve) as substrate, 5 ml of buffer, and the investigated acidifier. Initial pH was meas- 314 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation ured, and the vessel was flushed with CO2 before it was sealed with a rubber stopper and connected to an automatic gas production system. The vessels were incubated at 39°C, and gas production was measured every 15 min for 24 h. The magnetic stirrer mixed the vessel contents for 20 sec at inter- vals of 30 sec. At the end of the 24-h incubation, the fermentation fluid was sampled to determine its pH and its microbial metabolite concentrations. The fermentation fluid was frozen as such for lac- tic acid analysis according to Haacker et al. (1983). Volatile fatty acids were analysed according to Huhtanen et al. (1998), and for the analysis 1 ml of fermentation fluid was first mixed with 0.1 ml of saturated HgCl2, then with 0.4 ml of 1 N NaOH, and then frozen. For ammonia analysis, which was performed according to McCullough (1967), 1 ml of fermentation fluid was mixed with 20 µl of 50% H2SO4 and then frozen. In vitro fermentations were carried out in three consecutive steps. Firstly, the suitable acid addi- tion level for the culture medium was obtained by investigating the effects on the cumulative gas pro- duction resulting from adding 0, 5, 10, 20, or 30 µl of formic acid (100%) to 45 ml of culture medium. The respective formic acid concentrations were 2.9, 5.9, 11.8, and 17.7 mM, and there were two vessels for each formic acid level. Secondly, we compared the effects of different organic acids (formic, propionic, lactic, citric, and fumaric acid), organic salts (calcium formate, potassium sorbate, and sodium benzoate), and inorganic phosphoric acid by adding 20 µl of liquid or 20 mg of solid acidifiers to 45 ml of fermentation medium. There were at least three vessels per acid or salt. We end- ed up comparing different acidifiers on a volume/ weight-basis because additions producing a simi- lar molar concentration (12 mM) did not succeed. The molecular weights of the investigated acids and salts vary greatly, from 46 g mol-1 for formic acid to 192 g mol-1 for citric acid. Compared to the situation with formic acid, the amount in grams of the 12 mM additions of other acids and salts were 1.6–4.2 times higher, and consequently several of the additives completely stopped fermentation in the vessels. Furthermore, dietary organic acids are generally compared in vivo by adding acids to feed on the basis of weight rather than molar concentra- tion. In the third step, we studied the effects of mixing small amounts of sodium benzoate and/or potassium sorbate with formic acid. Both of these organic salts are known to be effective antimicro- bials (preservatives) in small dosages if combined with other acids that lower the pH (Chipley 1993, Sofos and Busta 1993). The investigated mixtures contained 50, 150, or 250 mg of sodium benzoate or potassium sorbate or 150 mg of both in 5 ml of formic acid (100%), resulting in mixtures contain- ing 1, 3, or 5% sodium benzoate or potassium sorbate or 2.5% of each. The addition level of these mixtures was 20 µl per 45 ml of fermentation me- dium, and there were three vessels per each acid treatment. Calculations and statistical analysis The proportion of undissociated acid in the fer- mentation medium at the initial pH level was cal- culated according to the Henderson-Hasselbach equation: pH = pKa + log[A -]/[HA]. A Gompertz bacterial growth model (Schofield et al. 1994) was adapted to the gas production data by using the NLIN procedure of SAS (1998). This model as- sumes that substrate limitations have no effect on growth, that the rate of growth is proportional to cell mass, and that the growth rate decays expo- nentially with time due to inactivation of the bac- teria. An alternative interpretation is that substrate levels limit growth in a logarithmic manner. The equation for gas production is as follows: V = VF exp {– exp [1 + (µme/VF)(λ – t)]} where, according to Zwietering et al. (1990), V = volume of gas produced at time t, t = fermentation time (h), VF = maximum volume of gas produced (ml), µm = maximum rate of gas production (ml h-1), which occurs at the point of inflection of the gas curve, and λ = the lag time (h), as the time axis intercept of a tangent line at the point of inflection. The duration of the exponential phase was calcu- lated from the parameters of the Gompertz func- tion as follows (Zwietering et al. 1992): 315 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. Exponential phase (h) = VF/(µme){1 – ln[(3 – √5 )/2]} The time required from the beginning of the fermentation to the maximum rate of gas produc- tion, i.e. from time 0 to the inflection point (TIP), was calculated as proposed by Piva et al. (2002): TIP (h) = λ + (exponential phase/2) The parameters estimated by means of the Gompertz function and the pH values and the mi- crobial metabolite concentrations in the fermenta- tion fluid were analysed with the MIXED proce- dure of SAS (1998) by using residual maximum likelihood estimation and having the fixed effect of treatment and the random effect of fermentation series in the model. When the F-test was signifi- cant (P < 0.05), differences between the treatments were identified by means of the Tukey test. Results The first step of fermentations with increasing for- mic acid concentrations (2.9–17.7 mM) showed that the Gompertz bacterial growth model nicely fitted (mean r2 = 0.997) microbial growth recorded using the cumulative gas production technique (Table 2). Gas production started almost immedi- ately, and the maximum rate of gas production (µm) differed from that of the control treatment at the level of 20 µl of formic acid per vessel, i.e. 11.8 mM (P < 0.05). At this addition level, the maximum volume of gas produced (VF) was about 89% of the amount produced in the control ves- sels. The length of the exponential phase did not differ at different formic acid levels (P > 0.05), but the time of the inflection point was earliest in ves- sels with the smallest formic acid addition (P < 0.05). Formic acid additions lowered the initial pH compared to the control treatment (P < 0.05). At the initial pH, the proportion of undissociated for- mic acid ranged from 2.2 to 5.0% with formic acid additions of 2.9 to 17.7 mM. At time zero, the fer- mentation fluid contained 62 mM lactic acid and 7.2 mM ammonia. After the 24-h fermentation, ammonia and lactic acid concentrations increased ca 2.6- and 2-fold compared to time zero, but they did not differ at different formic acid levels (P > 0.05). Because different acidifiers were compared on a volume/weight basis rather than a molar ba- Table 2. The effect of increasing formic acid additions (0, 5, 10, 20, or 30 µl per 45 ml) on the pH of the fermentation fluid (0 and 24 h), estimated parameters for the Gompertz bacterial growth model applied to the gas production data, and ammonia and lactic acid concentrations in the fermentation fluid after 24-h incubation (least-square means, n = 2). Formic acid, mM 0 2.9 5.9 11.8 17.7 SEM P Undissociated formic acid at pH0, mM 0.07 0.13 0.41 0.88 pH0 5.61 a 5.39b 5.39b 5.19c 5.03d 0.02 0.001 pH24 4.19 a 4.20a 4.15b 4.09c 4.05d 0.01 0.001 Coefficient of determination 0.998 0.998 0.997 0.998 0.996 Maximum volume of gas produced, ml 39.54 39.48 37.29 35.34 36.43 1.75 0.42 Maximum rate of gas production, ml h-1 9.06a 9.59a 9.54a 7.45b 7.46ab 0.43 0.05 Lag time, h 0.29 0.16 0.28 0.32 0.00 0.08 0.29 Exponential phase, h 3.16 2.97 2.83 3.45 3.53 0.21 0.30 Time from time 0 to inflection point, h 1.87bc 1.65a 1.70ab 2.05c 1.76abc 0.07 0.04 Lactic acid, mM 155 156 159 168 172 8 0.68 Ammonia, mM 15.8 14.5 15.9 14.1 13.5 0.7 0.22 a,b,c,d Within a row, means with different superscripts are significantly different according to the Tukey test (P < 0.05). 316 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation sis, molar concentrations of acid additions varied from 2.3 mM for citric acid to 11.8 mM for for- mic acid (Table 3). In all the vessels that con- tained acidifiers, the initial pH was lower than in the control treatment (P < 0.05), whereas no sig- nificant differences in final pH were detected. The calculated proportions of undissociated acid in vessels with different acids and salts were as follows: formic acid 1.4%, propionic acid 11.0%, lactic acid 1.0%, citric acid 0.2%, fumaric acid 0.8%, calcium formate 0.7%, sodium benzoate 1.8%, potassium sorbate 6.9%, and phosphoric acid 0.03%. Because the pH of the fermentation medium decreased during the fermentation, the concentrations of undissociated acids probably increased too. However, we do not know how fast the pH changed in the vessels. Based on the maximum rate of gas production, formic acid was the acidifier most effective in restricting mi- crobial fermentation in the vessels. In fact, it was the only acid that reduced the maximum rate of gas produced (µm) compared to the control treat- ment (P < 0.05). Furthermore, µm was slower in vessels with formic acid than in vessels with cal- cium formate, citric acid, and potassium sorbate (P < 0.05). Calcium formate addition increased µm compared to the control treatment (P < 0.05). The maximum volume of gas produced and the lag times did not differ between the treatments (P > 0.05). The length of the exponential phase ranged from 2.51 to 2.99 h, and the time of the inflection point from 1.53 to 1.77 h, but there were no significant differences between the acid treatments. At time zero, the fermentation fluid contained 44 mM lactic acid and 9.8 mM ammonia. During the 24-h fermentation, lactic acid concentration increased 1.6- to 3.2-fold and ammonia concen- trations about 1.5-fold, but the differences be- tween the acid treatments were not significant. Final ammonia concentrations were 16.0 mM in the control treatment and 14.5–15.1 mM in acid treatments, but differences were not significant. Final lactic acid concentration was 109 mM in the control treatment, and it ranged from 71 mM (calcium formate) to 139 mM (formic acid) in acid treatments. However, because of large varia- tion, the F-test for the treatment effect was not significant. Volatile fatty acids were analysed only in the following selected treatments: control, formic acid, lactic acid, sodium benzoate, and po- tassium sorbate (Table 4). At time zero, the fer- mentation fluid contained 12.0 mM acetic acid, 2.7 mM propionic acid, and 0.07 mM n-butyric acid. Formic acid, potassium sorbate, and sodium benzoate reduced total VFA, acetic acid, and pro- pionic acid concentrations compared to the con- trol treatment (P < 0.05). Furthermore, lactic acid addition resulted in larger total VFA, acetic acid, and propionic acid concentrations than formic acid or sodium benzoate did (P < 0.05). In addi- tion, the molar proportion of acetic acid was in- creased by formic acid and sodium benzoate ad- ditions compared with the control treatment (P < 0.05). The concentrations and molar proportions of other VFA were not influenced by these acidi- fiers (P > 0.05). The mixing of small amounts (1, 3, or 5%) of sodium benzoate or potassium sorbate with for- mic acid did not result in significant changes in gas production parameters compared to the re- sults with plain formic acid (Table 5). At time zero, the fermentation fluid contained 44.0 mM lactic acid, 9.8 mM ammonia, 12.4 mM acetic acid, 2.7 mM propionic acid, and 0.07 mM bu- tyric acid. Lactic acid and ammonia concentra- tions did not differ between the acid treatments (P > 0.05). Compared to the control treatment, the concentrations of acetic and n-butyric acids were reduced by all formic-acid-based mixtures (P < 0.05) but not by plain formic acid (P > 0.05). The results of the formic-acid-based mixtures did not differ from those of plain formic acid, with the exception of the acetic acid concentration in a mixture with 1% sodium benzoate (P < 0.05) and the n-butyric acid concentration in a mixture with 3% sodium benzoate (P < 0.05). The molar pro- portion of acetic acid was increased and that of propionic acid was decreased by all acid treat- ments (P < 0.05), but only the mixture with 3% sodium benzoate resulted in a larger proportion of acetic acid than that due to plain formic acid (P < 0.05). 317 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. T ab le 3 . T he e ff ec t of d if fe re nt o rg an ic a ci d an d sa lt a dd it io ns ( 20 µ l or m g pe r 45 m l) o n th e pH ( 0 an d 24 h ) of t he f er m en ta ti on fl ui d an d es ti m at ed p ar am et er s fo r th e G om pe rt z ba ct er ia l gr ow th m od el a pp li ed t o th e ga s pr od uc ti on d at a (l ea st -s qu ar e m ea ns ± s ta nd ar d er ro r of t he m ea n, n = 3 –6 ). A ci d or s al t ad di ti on U nd is so ci at ed ac id a t pH 01 pH 0 pH 24 C oe ffi ci en t of de te rm in at io n M ax im um v ol um e of g as p ro du ce d M ax im um r at e of ga s pr od uc ti on L ag t im e T re at m en t m M m M m l m l/ h h C on tr ol 6. 01 ± 0 .1 5e 4. 44 ± 0 .1 4 0. 99 2 48 .5 2 ± 5. 47 13 .1 3 ± 1. 34 bc 0. 33 ± 0 .2 2 F or m ic a ci d 11 .8 0. 17 5. 59 ± 0 .1 5a 4. 20 ± 0 .1 4 0. 99 1 42 .0 2 ± 5. 45 10 .4 9 ± 1. 33 a 0. 27 ± 0 .2 2 C al ci um f or m at e 3. 4 0. 02 4 5. 90 ± 0 .1 6c d 4. 70 ± 0 .1 6 0. 99 1 52 .7 1 ± 5. 87 15 .3 8 ± 1. 45 d 0. 27 ± 0 .2 2 P ro pi on ic a ci d 5. 9 0. 65 5. 79 ± 0 .1 6b c 4. 37 ± 0 .1 8 0. 99 3 44 .3 9 ± 6. 59 11 .6 6 ± 1. 59 ab 0. 33 ± 0 .2 3 L ac ti c ac id 5. 2 0. 05 4 5. 81 ± 0 .1 6b c 4. 36 ± 0 .1 6 0. 99 7 47 .1 4 ± 5. 97 11 .3 8 ± 1. 45 ab 0. 15 ± 0 .2 2 C it ri c ac id 2. 3 0. 00 5 5. 83 ± 0 .1 6c d 4. 32 ± 0 .1 8 0. 98 0 52 .0 8 ± 6. 59 15 .0 5 ± 1. 59 cd 0. 31 ± 0 .2 3 F um ar ic a ci d 3. 8 0. 03 1 5. 81 ± 0 .1 6c 4. 33 ± 0 .1 8 0. 99 0 46 .5 0 ± 6. 59 12 .1 4 ± 1. 59 ab 0. 31 ± 0 .2 3 P ot as si um s or ba te 3. 0 0. 20 5. 89 ± 0 .1 5c d 4. 37 ± 0 .1 4 0. 99 2 44 .0 1 ± 5. 45 12 .0 4 ± 1. 33 b 0. 41 ± 0 .2 2 S od iu m b en zo at e 3. 1 0. 05 4 5. 94 ± 0 .1 5d e 4. 34 ± 0 .1 4 0. 99 2 45 .5 7 ± 5. 54 11 .3 6 ± 1. 35 ab 0. 31 ± 0 .2 2 P ho sp ho ri c ac id 6. 6 0. 00 2 5. 68 ± 0 .1 6a b 4. 27 ± 0 .1 8 0. 99 2 43 .4 2 ± 6. 59 11 .6 4 ± 1. 59 ab 0. 31 ± 0 .2 3 P 0. 00 1 0. 09 0. 20 0. 00 1 0. 49 a, b , c , d , e W it hi n co lu m n, m ea ns w it h di ff er en t su pe rs cr ip ts a re s ig ni fi ca nt ly d if fe re nt a cc or di ng t o th e T uk ey t es t ( P < 0 .0 5) . 1 T he d eg re e of a ci d di ss oc ia ti on w as c al cu la te d on t he b as is o f th e H en de rs on -H as se lb ac h eq ua ti on . T ab le 4 . T he e ff ec t of s el ec te d or ga ni c ac id a nd s al t ad di ti on s (2 0 µl o r m g pe r 45 m l) o n am m on ia , l ac ti c ac id , a nd v ol at il e fa tt y ac id ( V FA ) co nc en tr at io ns i n th e fe rm en ta ti on fl ui d af te r 24 -h i nc ub at io n (l ea st -s qu ar e m ea ns ± s ta nd ar d er ro r of t he m ea n, n = 3 -6 ). T re at m en t C on tr ol F or m ic a ci d L ac ti c ac id P ot as si um s or ba te S od iu m b en zo at e P A ci d or s al t ad di ti on , m M 0 11 .8 5. 2 3. 0 3. 1 C on ce nt ra ti on , m M A m m on ia 16 .0 ± 1 .4 14 .6 ± 1 .4 14 .7 ± 1 .5 15 .2 ± 1 .4 14 .9 ± 1 .4 0. 35 L ac ti c ac id 10 8 ± 2 0 14 0 ± 1 9 13 0 ± 2 1 12 6 ± 1 9 12 6 ± 2 0 0. 18 T ot al V FA 52 .5 0± 8 .7 1d 40 .8 9 ± 8. 68 a 50 .9 3 ± 8. 78 cd 46 .8 3 ± 8. 68 bc 45 .4 1 ± 8. 68 b 0. 00 1 A ce ti c ac id 33 .1 3 ± 3. 78 d 27 .3 2 ± 3. 77 a 31 .8 6 ± 3. 80 d 29 .4 0 ± 3. 78 bc 29 .7 6 ± 3. 78 c 0. 00 1 P ro pi on ic a ci d 16 .5 4 ± 4. 06 d 12 .0 1 ± 4. 04 a 16 .1 7 ± 4. 09 cd 14 .1 4 ± 4. 04 bc 12 .9 6 ± 4. 04 ab 0. 00 2 n -B ut yr ic a ci d 1. 50 ± 0 .6 8 0. 84 ± 0 .6 5 1. 57 ± 0 .7 4 1. 74 ± 0 .6 5 1. 35 ± 0 .6 5 0. 41 I so bu ty ri c ac id 0. 11 ± 0 .0 2 0. 10 ± 0 .0 2 0. 09 ± 0 .0 2 0. 11 ± 0 .0 2 0. 11 ± 0 .0 2 0. 70 n -V al er ic a ci d 0. 85 ± 0 .7 6 0. 42 ± 0 .7 4 1. 05 ± 0 .8 1 1. 11 ± 0 .7 4 0. 91 ± 0 .7 4 0. 58 I so va le ri c ac id 0. 15 ± 0 .0 3 0. 13 ± 0 .0 3 0. 11 ± 0 .0 3 0. 13 ± 0 .0 3 0. 14 ± 0 .0 3 0. 57 C ap ro ni c ac id 0. 15 ± 0 .1 1 0. 07 ± 0 .1 1 0. 16 ± 0 .1 2 0. 20 ± 0 .1 1 0. 18 ± 0 .1 1 0. 52 C 2/ to ta l V FA 1 0. 64 ± 0 .0 5a 0. 69 ± 0 .0 5c 0. 66 ± 0 .0 5a bc 0. 65 ± 0 .0 5a b 0. 68 ± 0 .0 5b c 0. 04 C 3/ to ta l V FA 1 0. 31 ± 0 .0 3 0. 28 ± 0 .0 3 0. 30 ± 0 .0 3 0. 29 ± 0 .0 3 0. 27 ± 0 .0 3 0. 08 C 2/ C 31 2. 12 ± 0 .4 1 2. 64 ± 0 .4 0 2. 35 ± 0 .4 2 2. 35 ± 0 .4 0 2. 62 ± 0 .4 0 0. 06 a, b , c , d , e W it hi n co lu m n, m ea ns w it h su pe rs cr ip t ar e si gn ifi ca nt ly d if fe re nt a cc or di ng t o th e T uk ey t es t (P < 0 .0 5) . 1 T he r at io s of a ce ti c ac id t o to ta l V FA ( C 2/ to ta l V FA ), p ro pi on ic a ci d to t ot al V FA ( C 3/ to ta l V FA ), a nd a ce ti c to p ro pi on ic a ci d (C 2/ C 3) . 318 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation T ab le 5 . T he e ff ec t of f or m ic a ci d (F A ) an d fo rm ic -a ci d- ba se d bl en ds c on ta in in g so di um b en zo at e (N aB ) an d po ta ss iu m s or ba te ( K S ) on t he p H ( 0 an d 24 h ) of t he fe rm en ta ti on fl ui d, e st im at ed p ar am et er s fo r th e G om pe rt z ba ct er ia l gr ow th m od el , a nd m ic ro bi al m et ab ol it e co nc en tr at io n af te r 24 -h i nc ub at io n (l ea st s qu ar e m ea ns , n = 3 ). T re at m en t C on tr ol FA FA + 1% N aB FA + 3% N aB FA + 5% N aB FA + 1% K S FA + 3% K S FA + 5% K S FA + 2. 5% N aB an d K S S E M P A dd ed /u nd is so ci at ed ac id a t pH 0, m M FA 11 .8 /0 .1 2 11 .7 /0 .1 2 11 .4 /0 .1 1 11 .2 /0 .1 1 11 .7 /0 .1 1 11 .4 /0 .1 2 11 .2 /0 .1 1 11 .2 /0 .1 0 N aB – 0. 03 /0 .0 01 0. 09 /0 .0 02 0. 15 /0 .0 04 – – – 0. 07 /0 .0 02 K S – – – – 0. 03 /0 .0 03 0. 09 /0 .0 08 0. 15 /0 .0 13 0. 08 /0 .0 06 pH 0 6. 10 5. 72 5. 75 5. 78 5. 75 5. 76 5. 74 5. 77 5. 78 0. 06 0. 00 1 pH 24 4. 33 4. 26 4. 20 4. 17 4. 19 4. 19 4. 21 4. 21 4. 20 0. 08 0. 28 r2 0. 98 9 0. 98 9 0. 99 0 0. 98 8 0. 98 7 0. 98 6 0. 99 0 0. 98 4 0. 98 9 V F, m l 47 .9 4 73 .8 1 43 .1 9 45 .3 9 44 .0 4 44 .2 3 47 .1 2 40 .0 9 45 .0 7 6. 63 0. 35 µ m , m l h- 1 14 .9 3 11 .3 4 11 .5 3 11 .6 6 11 .3 1 11 .5 5 11 .8 6 11 .6 9 12 .5 0 0. 98 0. 15 λ, h 0. 38 0. 24 0. 26 0. 14 0. 26 0. 21 0. 13 0. 22 0. 19 0. 23 0. 35 C on ce nt ra ti on , m M A m m on ia 14 .9 15 .2 15 .0 12 .9 15 ,6 13 .3 14 .1 15 .0 14 .8 1. 5 0. 66 L ac ti c ac id 13 2 12 5 13 5 13 8 13 0 13 2 13 0 12 8 13 6 5. 1 0. 60 T ot al V FA 45 .5 3 39 .8 3 31 .4 7 32 .9 1 34 .3 6 34 .5 3 34 .5 0 35 .4 9 33 .9 4 5. 56 0. 06 A ce ti c ac id 28 .7 5c 25 .4 4b c 21 .3 3a 23 .0 6a b 23 .4 7a b 23 .3 7a b 22 .8 9a b 23 .9 1a b 22 .9 8a b 2. 52 0. 03 P ro pi on ic a ci d 15 .1 3 12 .8 0 9. 11 8. 94 9. 72 10 .0 9 10 .0 8 10 .4 8 9. 91 2. 71 0. 09 n- B ut yr ic a ci d 1. 04 c 0. 81 bc 0. 61 ab 0. 51 a 0. 65 ab 0. 59 ab 0. 57 ab 0. 58 ab 0. 57 ab 0. 15 0. 05 Is ob ut yr ic a ci d 0. 09 0. 09 0. 08 0. 08 0. 09 0. 07 0. 11 0. 09 0. 09 0. 02 0. 35 n- V al er ic a ci d 0. 31 0. 50 0. 16 0. 15 0. 24 0. 23 0. 20 0. 25 0. 19 0. 17 0. 32 Is ov al er ic a ci d 0. 13 0. 11 0. 12 0. 10 0. 11 0. 12 0. 12 0. 12 0. 13 0. 03 0. 50 C ap ro ni c ac id 0. 08 0. 08 0. 06 0. 06 0. 08 0. 06 0. 08 0. 07 0. 07 0. 02 0. 62 C 2/ to ta l V FA 2 0. 63 a 0. 67 b 0. 68 bc 0. 71 c 0. 69 bc 0. 69 bc 0. 68 bc 0. 69 bc 0. 68 bc 0. 03 0. 02 C 3/ to ta l V FA 2 0. 34 c 0. 30 b 0. 28 ab 0. 27 b 0. 28 ab 0. 28 ab 0. 29 ab 0. 29 ab 0. 29 ab 0. 03 0. 02 C 2/ C 31 1. 80 a 2. 38 b 2. 46 b 2. 71 b 2. 58 b 2. 51 b 2. 39 b 2. 49 b 2. 39 b 0. 37 0. 01 319 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. Discussion Organic acids are weak acids and therefore do not readily donate protons in aqueous solution. The relative strength of an acid is reflected in its dis- sociation constant Ka or its pKa value (-log Ka). In water, weak acids dissociate to a certain degree into protons and anions, and the dissociation is pH-dependent. Undissociated organic acids are li- pid soluble and can freely diffuse across the bacte- rial membrane into the cytoplasm, causing acidifi- cation of the cytoplasm. This influences microbial metabolism because the action of certain enzymes is inhibited, bacterial cells are forced to use energy to remove protons instead of taking up nutrients, and acid anions are accumulated in the cell (Cher- rington et al. 1991, Russell and Diez-Gonzalez 1998). In addition to being inhibitory agents, or- ganic acids can act as a carbon and energy source for micro-organisms. This however depends on the concentration of the acid, its ability to enter the cell, and the capacity of the organism to metabo- lize the acid (Cherrington et al. 1991). Organic salts have antimicrobial effects too. Calcium for- mate, sodium benzoate, and potassium sorbate are well soluble in water (Chipley 1993, Sofos and Busta 1993), and we therefore assume that they completely dissolved in the culture medium. In aqueous solutions, organic salts ionize easily, and the resulting formate, benzoate and sorbate anions can react with water to produce formic, benzoic, and sorbic acid, respectively, depending on the pH of the milieu and the pKa value of the acid. An in vitro gas production technique was used to investigate the ability of different organic acids (formic, propionic, lactic, citric, and fumaric acid), organic salts (calcium formate, potassium sorbate, and sodium benzoate), and inorganic phosphoric acid to modulate microbial fermentation in the di- gestive tract of piglets. Piva et al. (1999, 2002) used the gas production technique to study the ef- fects of organic acids on caecal fermentation. Ini- tially, our gas production system was set up by us- ing fresh ileal digesta from a growing pig. How- ever, the rate of fermentation varied greatly be- tween different fermentation series, probably be- cause of large variation in the composition and microbial activity of digesta collected on different days. It was therefore difficult to adjust the acid concentration in the vessels to a level at which it would influence fermentation but not completely inhibit it. To provide a more consistent culture me- dium and to ensure inoculum rich in organisms, we pooled digesta from several collection days and stored it in frozen form. Because freezing stops microbial acitivity in digesta, fermentation medium was inoculated with fresh faeces from suckling piglets that were consuming creep feed. A similar approach using frozen gastric and ileal di- gesta inoculated with piglet faeces in an in vitro fermentation system has been used by Knarreborg et al. (2002). We chose to use piglet faeces as in- oculum because dietary organic acid additions are primarily targeted towards piglets and because in piglets faeces are good indicators of general micro- biological changes occurring in the gastrointesti- nal tract (Canibe et al. 2001). The idea of in vitro gas production technique is to stimulate microbial fermentation occurring in the living digestive tract. However, there are sev- eral reasons which limit the direct application of these in vitro results to in vivo. The composition of ileal digesta from growing pig, which together with added feed served as substrate in the fermen- tation, differs from that of ileal digesta or faeces of young piglets (e.g. differences in diet composition, digestive capacity, and microflora). The initial pH was not adjusted to the same level, and it differed somewhat with different acid and salt additions. In vivo studies have shown that dietary organic acids may lower pH of gastric and duodenal contents but not further down in the gastrointestinal tract (Par- tanen and Mroz 1999, Mroz et al. 2001, Canibe et al. 2005). In the digestive tract of pig, both dietary organic acids as well as lactic acid and volatile fatty acids that are formed as a result of microbial fermentation are quickly absorbed and/or metabo- lized by intestinal microbes. Therefore, stomach and duodenum are considered as the primary sites of action of dietary organic acids (Partanen and Mroz 1999). Compared to in vivo, an in vitro batch fermentation system lacks the absorption and end- products of microbial fermentation accumulate in 320 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation the fermentation medium (Williams et al. 2005). Because no buffer was added to vessels during the 24-h incubation, the accumulation of microbial metabolites resulted in pH to decrease to al level which is lower than that generally found in the small and large intestinal contents of piglets (Canibe et al. 2001). The production of gas and microbial metabolites is dependent on the micro- bial population and available substrate in the fer- mentation medium. Heterofermentative lactic acid bacteria produce lactic acid and large amounts of carbon dioxide, whereas heterofermentative lactic acid bacteria produce primarily lactic acid but no gas. Short chain fatty acids, gaseous hydrogen and carbon dioxide are the primary products of hind gut fermentation (Ewing and Cole 1994). Gas is also released from buffer when it reacts with short chain fatty acids (Williams et al. 2005). Despite of these shortcomings, the in vitro gas production technique is a useful tool for primary screening of feed components (Williams et al. 2005) or dietary acidifiers (Piva et al. 1999) to find potential candi- dates for in vivo studies. Formic acid had the greatest effect on the max- imum rate of gas production, thereby exhibiting a greater inhibiting effect on microbial fermentation than the other acidifiers. Calcium formate differed from other acidifiers in that it stimulated microbial fermentation compared to the control treatment, as indicated by a higher maximum rate of gas produc- tion than in the control treatment. The effects of other acids did not differ from the control treat- ment. Piva et al. (1999) used the gas production technique and caecal digesta to evaluate the effects of different organic acids. They reported that 120– 240 mM formic and propionic acid decreased the maximum volume of gas produced by 29–55% 24–48% respectively, whereas acetic acid had no effect, and lactic acid increased gas production by 59–24%. The maximum rate of gas produced was slowed down by formic, acetic, and propionic ac- ids and increased by lactic acid. In the in vitro study by Knarreborg et al. (2002), benzoic acid was the most effective in inhibiting the growth of coliform bacteria in the stomach contents (pH 4.5), followed by fumaric, lactic, butyric, formic, and propionic acid, when the acids were used at a con- centration of 100 mM. In small intestinal contents (pH 5.5), the order of the effectiveness of the acids (100 mM) was somewhat different: benzoic acid > formic acid > butyric acid > propionic acid > lactic acid > fumaric acid. Lactic acid bacteria were able to grow in the presence of several acids both in the stomach and small intestinal contents, but their growth rates were reduced. Only fumaric acid and sodium benzoate exhibited strong antimicrobial properties towards lactic acid bacteria. The gastrointestinal tract of pigs hosts a diverse and dynamic community of bacteria, which re- sponds to dietary changes and infections (Leser et al. 2001). Yeasts are also found in the gastrointes- tinal tract of pigs (Canibe et al. 2001). It is reason- able to assume that the effects of different acidifi- ers on intestinal microflora differ. Formic acid is a strong acidulant that exhibits antimicrobial activity primarily against yeast and some bacteria such as Escherichia coli (Lueck 1980). Reduced lactic acid bacteria counts and/or lactic acid concentra- tions have been reported in the stomach and/or small intestine of pigs fed formic acid supplement- ed diets (Roth et al. 1992, Partanen et al. 2001a, Canibe et al. 2005). In the present study, the ves- sels with formic acid resulted in the highest con- centration of lactic acid after 24 h of fermentation. Lactic acid bacteria are able to grow at a low pH, and differences in initial pH may have influenced the fermentation pattern in the vessels. Final lactic acid concentration was negatively correlated with both initial (r = –0.61, P < 0.001) and final pH (r = –0.93, P < 0.001). Propionic acid is primarily ef- fective against moulds, but its activity against bac- teria is poor, and some yeast can metabolize it (Foeding and Busta 1991). Lactic acid is produced in the stomach and small intestine of pigs by sev- eral bacterial species, e.g. Lactobacillus, Bifido- bacterium, and Streptococcus. The antimicrobial activity of lactic acid is directed primarily against bacteria, whereas many yeast can metabolize it (Foeding and Busta 1991). Citric acid is generally a less effective antimicrobial agent because many micro-organisms can metabolize it (Foeding and Busta 1991). In this study, calcium formate stimu- lated microbial fermentation in the small intestine. Piva et al. (2002) reported an increased rate of gas 321 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 311–324. production when they used an acid mixture con- taining citric, malic, fumaric, and phosphoric ac- ids. Increased lactic acid production has been re- ported in the ileal contents of pigs fed diets sup- plemented with citric acid (Fasshauer and Kienzle 1995). Calcium formate contains ca 33% calcium (20 mg addition provided 6.6 mg calcium per 45 ml fermentation fluid), which may have acted as a buffer and higher pH has stimulated microbial fer- mentation. The lowest concentration of lactic acid observed in the calcium formate treatment indi- cates that a higher pH was not favourable for lactic acid producing bacteria. The growth-promoting effect of dietary organ- ic acids has been explained in part by decreased proteolysis and the decreased release of toxic sub- stances such as ammonia and amines (Kirchgess- ner and Roth 1988). Although, compared to the control treatment, all organic acid additions result- ed in numerically 6–10% lower concentrations of ammonia in the fermentation fluid after 24-h incu- bation, the differences between the results die to the different acids were not significant. The number of vessels would have had to be greater to be able show any possible significant effects. Piva et al. (2002) reported that an organic acid mixture con- taining phosphoric, citric, fumaric, and malic acid reduced ammonia concentration by about 13% in the caecal fermentation fluid. Organic acid also re- duced total VFA production and the ratio of acetic to propionic acid. In our study, VFA were deter- mined only in the control, formic acid, lactic acid, potassium sorbate and sodium benzoate treat- ments. Of these, formic acid addition resulted in the largest reduction in total VFA, acetic acid, and propionic acid concentrations. The effects of sodi- um benzoate and potassium sorbate were interme- diate between those of lactic and formic acids. Formic acid and potassium diformate additions have been shown to reduce ammonia and lactic acid concentrations in the stomach and small intes- tinal contents in vivo (Roth et al. 1992, Canibe et al. 2001, Partanen et al. 2001a). In vivo, dietary formic acid has been shown to increase total VFA and acetic acid concentrations in the small intesti- nal contents (Partanen et al. 2001a). The discrep- ancy between the in vitro and in vivo results could be due to the fact that lactic acid is quickly ab- sorbed and/or metabolized by microflora in the digestive tract of pigs. The second objective of this study was to in- vestigate the effects of adding small amounts of sodium benzoate and potassium sorbate to formic acid. We found that the addition of 5% potassium sorbate to formic acid was the most effective in reducing the maximum volume of gas produced (VF was 78% of the control treatment). The pKa value of sorbic acid is 4.76. The primary inhibitory activity of sorbic acid is against yeasts and moulds; the activity against bacteria is not as comprehen- sive and appears to be selective (Sofos and Busta 1993). Benzoic acid exhibits antimicrobial activity against bacteria, yeast and moulds (Chipley 1993). Ileal pig digesta contains yeasts, and it has been shown that their numbers decrease as a result of dietary additions of potassium diformate, formic acid, and benzoic acid but increase due to lactic acid additions (Maribo et al. 2000a, b, Canibe et al. 2001). Bacteria species inhibited by sorbate in- clude Escherichia, Lactobacillus, and others. The antimicrobial action of sorbate is pH dependent and increases when the pH approaches sorbate’s pKa value (4.76). Although the activity of sorbates is greater at low pH values, sorbates have the ad- vantage of being effective at pH values as high as 6.5–7.0. The maximum pH for most other com- mon preservatives is lower (Sofos and Busta 1993). Mixtures with sodium benzoate or potassium sorb- ate resulted in changes in total VFA, acetic, propi- onic, and n-butyric acid production. In perform- ance studies, dietary sorbic acid additions (1.2– 2.4%) have had profound growth-promoting ef- fects (14–27 % increase in growth rate) in weaned piglets with a body weight between 7 and 26 kg (Kirchgessner et al. 1995). Maribo et al. (2000b) reported that 2.0 and 1.0% benzoic acid in starter and weaner diets enhanced the growth rate of pig- lets aged 4–6 and 6–10 weeks by 18 and 16%, re- spectively. Jørgenssen and Boes found that an ad- dition of 1.0% benzoic acid to diets increased the growth rate of piglets weighing 9–32 kg by ca 10%. In conclusion, this in vitro study shows that or- ganic acids and salts used as growth promoters in 322 A G R I C U L T U R A L A N D F O O D S C I E N C E Partanen, K. & Jalava, T. Effects of organic acids and salts on swine microbial fermentation pig diets differ in their ability to modulate micro- bial fermentation in the digestive tract of piglets. Formic acid was the acidifier most effective in re- stricting microbial fermentation in a culture medi- um that contained ileal digesta and feed as sub- strate and faecal inoculum from piglets. Use of mixtures of formic acid with a small amount of sorbate or benzoate changed fermentation profiles more than the use of plain formic acid. Use of mix- tures of formic acid with potassium sorbate or so- dium benzoate altered microbial metabolite con- centrations more than use of plain formic acid. The effects of these mixtures should therefore be inves- tigated further in future in vivo studies. Acknowledgements. 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Effects of organic acids and salts on swine microbial fermentation SELOSTUS Orgaanisten happojen ja suolojen vaikutus mikrobifermentaatioon porsaan ruuansulatuskanavassa in vitro Kirsi Partanen ja Taina Jalava MTT (Maa- ja elintarviketalouden tutkimuskeskus) In vitro -kaasuntuotantomenetelmällä selvitettiin erilais- ten orgaanisten happojen (muurahais-, propioni-. maito-, sitruuna- ja fumaarihappo) ja suolojen (kalsiumfor- miaatti, kaliumsorbaatti ja natriumbentsoaatti) sekä epä- orgaanisen fosforihapon mahdollisuutta muokata mikro- bifermentaatiota porsaan ruoansulatuskanavassa. Lasi- pulloihin laitettiin inkubaatiota varten 40 ml seosta, jossa oli 53 % puskuria, 45 % ohutsuolen ruokasulaa ja 2 % sontaa, sekä 5 ml puskuria, 0,5 g rehua ja 20 µl nes- temäisiä tai 20 mg kiinteitä happoja ja suoloja. Kaasun- tuotanto mitattiin automaattisesti 15 minuutin välein seuraavan 24 h ajan. Muurahaishappo oli ainoa, joka hi- dasti maksimaalista kaasun muodostumisnopeutta kont- rolliin verrattua. Muurahaishappoa sisältäneissä pullois- sa kaasua muodostui hitaammin kuin kalsiumformiaat- tia, sitruunahappoa ja kaliumsorbaattia sisältäneissä pul- loissa. Kalsiumformiaatti nopeutti kaasun muodostu- mista kontrolliin verrattuna. Muodostuneen kaasun ko- konaismäärässä ei ollut merkitseviä eroja happojen ja suolojen välillä. Kun tutkimme muurahaishappopohjai- sia seoksia, joissa oli 1–5 % natriumbentsoaattia ja/tai kaliumsorbaatia, kaasun muodostumista kuvaavat para- metrit eivät eronneet puhtaan muurahaishapon vaikutuk- sesta. Kaikki muurahaishappopohjaiset seokset pienen- sivät haihtuvien rasvahappojen, etikkahapon, propioni- hapon ja n-voihapon pitoisuutta fermentaationesteessä, mutta ei pelkkä muurahaishappo. Johtopäätöksenä on, että orgaanisilla hapoilla ja niiden suoloilla on erilainen kyky muokata mikrobifermentaatiota porsaan ruuansu- latuskanavassa. Lisäämällä natriumbentsoaattia tai ka- liumsorbaattia muurahaishappoon voidaan muuttaa fer- mentaation tyyppiä. Effects of some organic acids and salts on microbialfermentation in the digestive tract of pigletsestimated using an in vitro gas production technique Introduction Material and methods Results Discussion References SELOSTUS