Evaluating the potential of lignosulfonates and chitosans as alfalfa hay preservatives using in vitro techniques.

Our objectives were to compare the antifungal activity of 5 lignosulfonates, and 2 chitosans against fungi isolated from spoiled hay, and assess the effects of an optimized lignosulfonate, chitosan, and propionic acid (PRP) on high-moisture alfalfa hay. In experiment 1, we determined the minimum inhibitory concentration and minimum fungicidal concentration of 4 sodium lignosulfonates, 1 magnesium lignosulfonate, 2 chitosans, and PRP (positive control) against Aspergillus amoenus, Mucor circinelloides, Penicillium solitum, and Debaromyces hansenii at pH 4 and 6. Among sodium lignosulfonates, the one from Sappi Ltd. (NaSP) was the most antifungal at pH 4. However, chitosans had the strongest fungicidal activity with the exception of M. circinelloides at both pH 4 and 6. PRP had more antifungal effects than NaSP and was only better than chitosans for M. circinelloides. In experiment 2, we evaluated the effects of 3 additives (ADV): optimized NaSP (NaSP-O, UMaine), naïve chitosan (ChNv, Sigma-Aldrich), and PRP on high-moisture alfalfa hay. The experimental design was a randomized complete block design replicated 5 times. Treatment design was the factorial combination of 3 ADV× 5 doses (0, 0.25, 0.5, 1, and 2% w/w fresh basis). Additives were added to 35 g of sterile alfalfa hay (71.5 ± 0.23% DM), inoculated with a mixture of previously isolated spoilage fungi (5.8 log cfu/fresh g), and aerobically incubated in vitro for 23 d (25°C). After incubation, DM losses were reduced by doses as low as 0.25% for both NaSP-O and PRP (x¯=1.61) vs. untreated hay (24.0%), partially due to the decrease of mold and yeast counts as their doses increased. Also, hay NH3-N was lower in NaSP-O and PRP, with doses as low as 0.25%, relative to untreated hay (x¯=1.13 vs. 7.80% of N, respectively). Both NaSP-O and PRP increased digestible DM recovery (x¯=69.7) and total volatile fatty acids (x¯=94.3), with doses as low as 0.25%, compared with untreated hay (52.7% and 83.8 mM, respectively). However, ChNv did not decrease mold nor yeast counts (x¯=6.59 and x¯=6.16 log cfu/fresh g, respectively) and did not prevent DM losses relative to untreated hay. Overall, when using an alfalfa hay substrate in vitro, NaSP-O was able to prevent fungal spoilage to a similar extent to PRP. Thus, further studies are warranted to develop NaSP-O as a hay preservative under field conditions.

In our first experiment, we assessed the antifungal activity of two major types of byproducts, one known as lignosulfonates (5 types), which are generated by paper mills, and another known as chitosans (2 types), which are generated from shellfish. These were tested against four fungi isolated from spoiled hay. We observed that acidic conditions are not necessary for chitosans but are crucial to activate the antifungal properties of lignosulfonates. Also, we found that sodium lignosulfonate from Sappi Ltd. was the most antifungal relative to other sodium lignosulfonates from other manufacturers. Chitosans had stronger fungicidal activity than propionic acid or lignosulfonates against all but one mold tested. In our second experiment, we compared the best treatments from experiment 1 against propionic acid using alfalfa hay as a substrate to grow the same fungi tested in experiment 1. None of the doses of chitosan prevented spoilage on high moisture hay, showing results similar to untreated hay. In contrast, an optimized sodium lignosulfonate and propionic acid prevented fungal spoilage of alfalfa hay with doses as low as 0.25%.

Effect of chemical and biological preservatives and ensiling stage on the dry matter loss, nutritional value, microbial counts, and ruminal in vitro gas production kinetics of wet brewer's grain silage.

This study evaluated the effects of chemical and biological preservatives and ensiling stage on spoilage, ruminal in vitro fermentation, and methane production of wet brewer's grain (WBG) silage. Treatments (TRT) were sodium lignosulfonate at 10 g/kg fresh WBG (NaL1) and 20 g/kg (NaL2), propionic acid at 5 g/kg fresh WBG (PRP, 99%), a combination inoculant (INO; Lactococcus lactis and Lactobacillus buchneri each at 4.9 log cfu per fresh WBG g), and untreated WBG (CON). Fresh WBG was treated and then ensiled for 60 d, after which mini silos were opened and aerobically exposed (AES) for 10 d. Data were analyzed as an RCBD (five blocks) with a 5 TRT × 3 stages (STG; fresh, ensiled, and AES) factorial arrangement. Results showed that ensiled PRP-treated WBG markedly preserved more water-soluble carbohydrates and starch than all other ensiled TRT (P < 0.001). Dry matter losses of ensiled PRP-treated WBG were 48% lower than all other ensiled TRT (P = 0.009) but were not different than CON in AES (P = 0.350). Due to its greater concentration of digestible nutrients, PRP-treated AES was less aerobically stable than CON (P = 0.03). Preservation was not improved by INO, NaL1, or NaL2 but the latter prevented the increase of neutral detergent fiber across STG (P = 0.392). Apparent in vitro DM digestibility (IVDMD) decreased only in ensiled CON, INO, and NaL1 relative to fresh WBG and AES NaL2 had greater IVDMD than all other AES TRT (P ≤ 0.032). In vitro ruminal fermentation of fresh WBG resulted in a greater methane concentration and yield than the other STG (P < 0.033). In conclusion, PRP was the most effective at preserving WBG during ensiling but failed to improve aerobic stability under the conditions tested.

Wet brewer's grain (WBG) is the most abundant byproduct in the manufacture of beer and its rich nutritional composition makes it a valuable feed for cattle. However, WBG is highly susceptible to spoilage so the application of cost-effective preservatives may be a viable approach to prevent nutrient losses during ensiling and feed out. The present study evaluated the effects of chemical and biological preservatives on the nutritional composition and in vitro fermentation and gas production of WBG across three silage production stages: fresh, ensiled, and aerobically exposed silage (AES). Preservatives tested were propionic acid, a bacterial inoculant, and sodium lignosulfonate (NaL) applied at 1% and 2%. Propionic acid successfully reduced the loss of nutrients and preserved more sugars and starch than all other treatments during ensiling, which resulted in higher digestibility in vitro. However, due to its greater concentration of digestible nutrients, ensiled WBG treated with propionic acid also suffered extensive spoilage in the AES. All other treatments failed to improve the preservation of ensiled or AES WBG, but NaL at 2% prevented the decrease of digestibility for AES.

Meta-analysis of the effects of chemical and microbial preservatives on hay spoilage during storage.

A meta-analysis was performed to evaluate the effects of chemical (50 articles) and microbial (21 articles) additives on hay preservation during storage. Multilevel linear mixed-effects models were fit with response variables calculated as predicted differences (Δ) between treated and untreated samples. Chemical preservatives were classified into five groups such as propionic acid (PropA), buffered organic acids (BOA), other organic acids (OOA), urea, and anhydrous ammonia (AA). Moderators of the models included preservative class (PC), forage type (FT; grass, legumes, and mixed hay), moisture concentration (MC), and application rate (AR). Dry matter (DM) loss during storage was affected by PC × FT (P = 0.045), PC × AR (P < 0.001), and PC × MC (P = 0.009), relative to the overall effect of preservatives (-0.37%). DM loss in PropA-treated hay was numerically reduced to a greater extent in grasses (-16.2), followed by mixed hay (-1.76), but it increased (+2.2%) in legume hay. Increasing AR of PropA resulted in decrease in DM loss (slope = -1.34). Application of BOA, OOA, PropA, and AA decreased visual relative moldiness by -22.1, -29.4, -45.5, and -12.2 percentage points, respectively (PC; P < 0.001). Sugars were higher in treated grass hay (+1.9) and lower in treated legume hay (-0.8% of DM) relative to their untreated counterparts (P < 0.001). The application of all preservatives resulted in higher crude protein (CP) than untreated hay, particularly urea (+7.92) and AA (+5.66% of DM), but PropA, OOA, and BOA also increased CP by 2.37, 2.04, and 0.73 percentage points, respectively. Additionally, preservative application overall resulted in higher in vitro DM digestibility (+1.9% of DM) relative to the untreated hay (x¯=58.3%), which increased with higher AR (slope = 1.64) and decreased with higher MC (slope = -0.27). Microbial inoculants had small effects on hay spoilage because the overall DM loss effect size was -0.21%. Relative to untreated (x¯=4.63% DM), grass hay preserved more sugars (+1.47) than legumes (+0.33) when an inoculant was applied. In conclusion, organic acid-based preservatives prevent spoilage of hay during storage, but their effectiveness is affected by FT, MC, and AR. Microbial inoculants had minor effects on preservation that were impaired by increased MC. Moreover, legume hay was less responsive to the effects of preservatives than grass hay.

Storing hay that has not been properly dried to a moisture concentration (MC) below 20% can lead to the growth of undesirable microbes, such as molds, that decrease the nutritive value of the hay and compromise animal performance and health. Hay preservatives such as propionic acid (PropA), buffered organic acids (BOA), other organic acids (OOA), urea, anhydrous ammonia (AA), and microbial inoculants have been commonly applied to high moisture (>20%) hay to reduce microbial growth during hay storage. The present study compiled the results of 62 published articles on hay preservatives and performed a quantitative analysis (meta-analysis) to determine the effect of preservative treatment on dry matter (DM) loss, moldiness, bale heating, nutritional composition, and DM digestibility, and their interactions with forage type, application rate, and bale moisture. Organic acid-based preservatives (PropA, BOA, and OOA) were effective at reducing DM loss, moldiness, bale maximum temperature, and insoluble nitrogen, and at preserving sugars and DM digestibility during storage to different extents. Microbial inoculants had small effects on the prevention of hay spoilage, and were negatively affected by the increase of hay MC. Legume hay was less responsive to the effects of preservatives than grass hay during storage.

A combination of Lactobacillus buchneri and Pediococcus pentosaceus extended the aerobic stability of conventional and brown midrib mutants-corn hybrids ensiled at low dry matter concentrations by causing a major shift in their bacterial and fungal community.

We evaluated the effects of applying a combination inoculant to four corn hybrids harvested at high moisture on their nutritive value and microbial populations. The treatment design was the factorial combination of corn hybrids ensiled with (INO) and without (CON) inoculant. The hybrids were TMF2R737 (MCN), F2F817 (MBR), P2089YHR (PCN), and PI144XR (PBR), ensiled at dry matter (DM) concentrations of 30.5%, 26.3%, 31.1%, and 31.5%, respectively; MBR and PBR were brown midrib mutants (BMR). The inoculant contained Lactobacillus buchneri and Pediococcus pentosaceus (4 × 105 and 1 × 105 cfu/g of fresh corn). The experiment had a complete randomized design with treatments replicated six times. Corn was treated or not with inoculant, packed into 7.6 L bucket silos, and stored for 100 d. At d 0, the relative abundance (RA, %) of Enterobacteriaceae was lower in PBR vs. the other hybrids [51.3 vs. x¯ = (average of) 58.4] and in the case of fungi, incertae sedis (i.s.) Tremellales and Mucoraceae were more and less abundant, respectively, in conventional hybrids vs. BMRs (x¯= 25.8 vs. x¯ = 13.9 and x¯ = 3.64 vs. x¯ = 7.52; P < 0.04). After ensiling, INO had higher LAB (9.3 vs. 7.1 log cfu/g of fresh corn) and acetic acid (3.44% vs. 1.32% of DM) and lower yeast (3.1 vs. 4.6) and molds (1.5 vs. 3.0), and also extended the aerobic stability (582 vs. 111 h) but decreased DM recovery (95.6% vs. 97.4%) vs. CON (P < 0.02). Inoculation reduced bacterial phylogenetic diversity (6.75 vs. 14.4) but increased fungal observed taxonomical units (46 vs. 20) vs. CON (P < 0.01). Also, a higher relative abundance (RA) for Lactobacillaceae (99.2% vs. 75.7%) and lower for Enterobacteriaceae (0.28 vs. 9.93) was observed due to inoculation (P < 0.001). For fungi, INO had a lower RA compared to CON for Monascaceae (12.6 vs. 44.7) and increased i.s. Tremellales (8.0 vs. 1.2) and i.s. Saccharomycetales (6.4% vs. 0.3%; P < 0.006). Inoculation changed the diverse bacterial community found in the phyllosphere across hybrids to a taxonomically uneven one dominated by Lactobacillaceae. In the case of fungi, INO application increased the fungal diversity at d 100 mainly by reducing the dominance of Monascaceae vs. CON. In conclusion, the INO treatment overwhelmed the disparate microbial populations found across BMR and conventional hybrids ensiled at low DM concentrations and ensured a significant concentration of acetic acid that modified fungal populations and in turn extended the aerobic stability of all hybrids.