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

Abstract

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.

Keywords: chitosan; hay preservation; lignosulfonates; spoilage.

Figure 1.

Fourier transform infrared spectra of lignosulfonates. NaSP, sodium lignosulfonate (Sappi North America, Boston, MA); MgSP, magnesium lignosulfonate (Sappi North America, Boston, MA); NaAL, lignosulfonic acid sodium salt (Sigma–Aldrich Corp, St Louis, MO); NaUM, sodium lignosulfonate (Spectrum Chemical MFG Corp, New Brunswick, NJ); and NaBT, lignosulfonic acid sodium salt (BeanTown Chemical, Hudson, NH).

Figure 2.

Flowchart for the in vitro aerobic evaluation method of hay spoilage (modified from Reyes et al., 2020).

Figure 3.

Dry matter loss (%) of ground alfalfa hay as a function of additive and dose after a 23-d in vitro aerobic incubation. NaSP-O, optimized lignosulfonate-based product; ChNv, naïve chitosan; PRP, propionic acid. Uppercase letters depict differences across ADV within dose (P ≤ 0.05) and lowercase letters depict differences across dose within ADV (PRP: x, y and NaSP-O: m, n; P ≤ 0.05; SEM = 0.451).