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Article

In Vitro Fermentation Characteristics of Pine Needles (Pinus densiflora) as Feed Additive

by
Young-A Hwang
1,†,
Woo-Do Lee
1,2,†,
Juhyeon Kim
1,
Solhee Kim
1,
Min-Gyung Choi
1,
Jeong-Yeon On
1,
Sang-Woo Jeon
1,
Sung-Gu Han
3 and
Soo-Ki Kim
1,*
1
Department of Animal Science and Technology, Konkuk University, Seoul 05029, Republic of Korea
2
Poultry Research Institute, National Institute of Animal Science, RDA, Pyeongchang 25342, Republic of Korea
3
Department of Food Science and Biotechnology of Animal Resource, Konkuk University, Seoul 05029, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(5), 415; https://doi.org/10.3390/fermentation9050415
Submission received: 30 March 2023 / Revised: 23 April 2023 / Accepted: 24 April 2023 / Published: 26 April 2023
(This article belongs to the Section Probiotic Strains and Fermentation)
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Abstract

:
In this study, the fermentation characteristics of pine needles were investigated for the first time and the possibility of using them as plant-derived feed additives was confirmed. Four types of fermented pine needle were prepared: (1) natural fermentation (CON); (2) Lactobacillus plantarum SK4315 fermentation (LPF); (3) Saccharomyces cerevisiae SK3587 fermentation (SCF); and (4) co-culture fermentation (CCF). Fermentation lasted 48 h, and samples were taken at 4-h intervals until 12 h, then at 24 and 48 h. As analysis items, fermentation characteristics, antibacterial, antioxidant, and enzymatic activities were investigated. As a result, all pine needle cultures were fermented with changes in the number of viable cells and pH. LPF inhibited the growth of the most pathogens, and the activity became stronger with fermentation. Total polyphenol content (TPC) was the highest in the 48 h SCF and LPF samples, ABTS radical scavenging ability and intracellular antioxidant activity were higher in SCF. Enzymatic activities were different depending on each pine needle culture medium and the fermentation time. In summary, all pine needle cultures were normally fermented, and as fermentation progressed, LPF strengthened antibacterial activity and SCF strengthened antioxidant activity. This study confirmed the potential of fermented pine needles as a feed additive, showing an enhancement of biological activity.

1. Introduction

As the use of antibiotics in feed has recently been banned in the animal industry, studies have been conducted on the functionality and additive effects of substances such as probiotics, prebiotics, synbiotics, amino acids, vitamins, and enzymes [1,2,3,4]. Among them, probiotics are natural feed additives that have been used for a long time, and lactic acid bacteria (LAB), Bifidobacterium spp., and yeast (Saccharomyces boulardii, S. cerevisiae etc.) are useful microorganisms the stability of which has been recognized [5,6]. The intervention of probiotics provides health benefits to the host by preventing disease and enhancing immunity through the increase of beneficial intestinal-microorganisms and inhibiting intestinal-pathogenic bacteria [7]. In addition, it has been reported that the use of probiotics increases productivity, such as feed intake, feed conversion ratio, and daily weight gain [8,9,10,11], and is effective in improving the quality of animal products [12,13]. On the other hand, the probiotics complex is known to have a more positive synergistic effect than using a single strain [14,15,16], but the possibility of using it has not been well verified [16,17].
More than 100 species of pine tree (Pinus sp.) belonging to the conifers are known worldwide [18]. In Korea, pine trees account for around 26% of the forest ecosystem, and representative species include red pine (P. densiflora S. et Z.), pitch pines (P. rigida Mill), Keumkang pines (P. densiflora for. erecta), and sea pines (P. thunbergii) [19,20]. A substantial number of secondary metabolites such as terpenoids, phenolics, cinnamic acids, carboxylic acids, fatty acids, and flavonoids are present in pine needles [21]. These metabolites are known to have positive effects on physical health, such as anti-inflammatory, anti-microbial, anti-neurodegenerative, anti-tumoral, and cardioprotective [22]. For this reason, pine needles are used as food additives, functional foods, and pharmaceuticals. [22]. Nevertheless, the reuse of pine byproducts (pine needles, shoots, bark, etc.) is still limited compared to its potential value, and more research is needed to find new products and applications [18].
Plants contain anti-nutritional factors, macronutrients, micronutrients, and phytochemicals [23]. There are several ways to improve plant nutrient availability, one of which is fermentation using probiotics [23]. Fermentation is a way to prevent food from spoiling and helps to improve the flavor, digestibility, nutritional content, and palatability of the substrate; in particular, it can enhance its biochemical properties, such as antioxidant and antibacterial activity [24]. Chinese chives (Allium tuberosum) fermentation through Lactobacillus plantarum improved functional substances such as propanoic acid, benzoylmesaconine, kaempferol, isorhamnetin, quercetin, hydroxystearic acid, and saponins in Chinese chives, and improved their ability to inhibit the avian influenza virus and pathogens [25]. For this reason, studies on a comparison of physiological activity before and after the fermentation of plants and applications to livestock (pigs, poultry, daily cows, etc.) as a natural fermented feed additive have been conducted [24,26,27,28].
In other words, pine needles have various physiological substances, and fermentation through useful microorganisms improves the functionality of natural products, but research on the characteristics of fermented pine needles and the possibility of using fermented pine needles as products is still insignificant. Therefore, in this study, we investigated the applicability as a fermented feed additive using pine needles, a byproduct of P. densiflora, one of the representative pine trees in Korea. Fermenting microorganisms, L. plantarum and/or S. cerevisiae, which are widely used because their stability has been confirmed, were used, and an effective livestock feed additive was selected by comparing the antibacterial, antioxidant, and enzymatic activities of each fermented pine needle. The development of fermented pine needle feed additives in this study is expected to help improve the breeding environment, such as maintaining the health of livestock.

2. Materials and Methods

2.1. Pine Needles and Microorganisms for Fermentation

The pine needles used in this study were thinned Keumkang pine (P. densiflora; Bonghwa, Gyeongsangbuk-do, Republic of Korea). After washing with tap water three times, it was naturally dried at room temperature (22 ± 2 °C) for 3 days, and the dried pine needle was cut into 1–2 cm. Then, samples were stored in a freezer at −20 °C. The results of the nutrient component analysis of the pine needles are shown in Table 1 [29]. In addition, P. densiflora pine needles are known to have 370 mg/g of lignin, 407 mg/g of total carbohydrate content, 57.71 mg/g of total polyphenol content, and 14.99 mg/g of total flavonoid content [30,31].
As microorganisms for fermentation, L. plantarum SK4315 and S. cerevisiae SK3587 from our laboratory were used. S. cerevisiae SK3587 was cultured in Yeast Malt (YM) broth at 30 °C for 24 h, and L. plantarum SK4315 was cultured in De Man, Rogosa, and Sharpe (MRS) broth at 37 °C for 24 h.

2.2. Fermentation of Pine Needles and Changes in Viable Cell Count and pH

Changes in pH and viable cell count according to pine needle fermentation were investigated for 48 h. A total of 4 types of fermented pine needle were prepared: natural fermentation (no probiotic inoculation; Control; CON), L. plantarum SK4315 fermentation (LPF), S. cerevisiae SK3587 fermentation (SCF), and co-culture fermentation (CCF). All culture medium used pine needles, molasses, and distilled water at a ratio of 2:1:7, respectively, and the starting viable cell count was inoculated so that it was 106 colony forming units (CFU)/mL or more. For the fermentation conditions, CON and LPF were cultured at 37 °C, SCF and CCF were cultured at 30 °C, and the agitation of all cultures was maintained at 100 rpm. The culture solution for each treatment was designed in triplicate.
The number of viable cells was measured by serial dilution, and YM, MRS, Luria-Berani (LB) agar plates were used. The pH was measured using a pH meter (ISTEK 735P, Seoul, Korea). Analysis was measured at 4-h intervals from 0 h to 12 h, and then measured at 24 and 48 h to observe changes.

2.3. Antibacterial Activity Using Agar Well-Diffusion Assay

The antibacterial activity of each fermentation broth of pine needles against pathogenic microorganisms was evaluated. A total of 10 microorganisms were prepared as pathogens, including intestinal and respiratory pathogens and food poisoning bacteria: Listeria monocytogenes, Clostridium perfringens, Enterotoxigenic Escherichia coli, Staphylococcus aureus, Salmonella gallinarum, Escherichia coli, Burkholderia contaminans., Haemopillus parasuis, Haemopillus somnus, and Pantoea agglomerans. Each pathogen was cultured in LB broth for 24 h at 100 rpm, and 100 µL of the pathogen cultures were swabbed on an LB agar plate. Then, each fermentation broth was added into the well (diameter 6 mm) on each agar plate and incubated at 37 °C for 18 h, and the clear zone of inhibition (mm) was measured. Antibacterial activity was compared and analyzed using 24 and 48 h fermentation samples.

2.4. Determination of Total Phenols and Flavonoids

The total polyphenol content (TPC) and total flavonoid content (TFC) were referred to the method of Kwon et al. [32]. Briefly, 100 µL each of the sample and the 1N Folin-Ciocalteu solution were mixed and reacted at room temperature for 3 min. Then, 200 µL of 1N Na2CO3 solution was mixed and reacted at room temperature in the dark for 90 min. After the reaction, absorbance was measured at 725 nm by enzyme-linked immunosorbent assay (ELISA; Synergy 2, BioTek Instruments Inc., Winooski, VT, USA). Gallic acid (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as standard, and TPC was expressed as gallic acid equivalent (GAE) micrograms per mL of fermented pine needle culture.
For the total flavonoid content, 0.42 mL of the sample, 2.1 mL of diethylene glycol, and 0.21 mL of 1N NaOH were mixed and reacted in a water bath at 37 °C for 1 h. After the reaction, absorbance was measured at 420 nm through ELISA. Quercetin (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as standard, and TFC was expressed as a microgram of quercetin equivalent (QE) per mL of fermented pine needle culture.

2.5. Antioxidant Assays

The antioxidant activity of each fermented product was investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) [32].
For the measurement of DPPH radical scavenging activity, 100 μL of the sample, 400 μL of distilled water, and 2 mL of 0.15 mM DPPH solution (Sigma-Aldrich Corp., St. Louis, MO, USA) were mixed and reacted for 15 min at room temperature in the dark. Ascorbic acid was used as a standard, the percentage of the radical scavenging effect (%) was calculated by measuring the absorbance at 734 nm. The formula used is as follows:
Radical   scavenging   activity   ( % ) = A c − A s A c   × 100;
Ac: Control absorbance; As: Sample absorbance
For ABTS analysis, two solutions of 14.8 mM ABTS (Sigma-Aldrich Corp., St. Louis, MO, USA) and 5.0 mM potassium persulfate (Sigma-Aldrich Corp., St. Louis, MO, USA) were mixed in equal amounts and reacted in a dark room at room temperature for 16 h. Thereafter, the resulting solution was diluted with water until an absorbance of 0.70 at 734 nm was reached. The 180 μL of the diluted ABTS solution was mixed with 20 μL of the fermented sample and reacted for 30 min at room temperature in the dark. The percentage of radical scavenging effect (%) was calculated using the formula above. For comparison of the DPPH radical scavenging ability, samples fermented for 0, 4, 8, 12, 24, and 48 h were used. ABTS measured samples fermented for 48 h in consideration of DPPH radical scavenging ability, and in particular, the fermentation effect was observed by simultaneous comparison of non-fermented (NF) samples not inoculated with fermenting microorganisms.

2.6. Superoxide Radical-Scavenging Assay

Each fermented pine needle culture was centrifuged at 13,000 rpm for 15 min in a centrifuge (Mega 18R, Hanil Science Co., Ltd., Deajeon, Republic of Korea) to separate the supernatant, and then filtered using filter paper (No.1 qualitative filter paper, Advantec, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and a syringe filter (Minisart® syringe filter 0.45 μm, Sartorius, Germany). For porcine aortic endothelial (PAE) cell culture, M199 medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin was used and cultured for 72 h in a humidified incubator at 5% CO2 and 37 °C. PAE cells were pretreated with 50 μL of the fermentation sample for 16 h, and then treated with E. coli O111:B4-derived lipopolysaccharide (LPS) at a concentration of 1 μL/mL for 4 h. Then, the cells were treated with the fluorescent dye dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich Corp., St. Louis, MO, USA) at a concentration of 10 μM for 30 min, and then washed twice with 1× phosphate-buffered saline (PBS). Then, H2O2-induced cells were confirmed using a fluorescence microscope (IX71, Olympus, PA, USA), a microscope (DP71, Olympus, PA, USA), and DP controller software (Olympus Optical Co. Ltd., Tokyo, Japan). For quantification, fluorescence intensity was measured with ImageJ software (National Institute of Health, Bethesda, MD, USA).

2.7. Enzymatic Activities

Referring to the method of Niu et al. [33], a medium containing each substrate was prepared to evaluate the enzymatic activities of amylase, cellulase, protease, lipase, and xylanase of each fermented pine needle culture medium. As the basal medium, LB, YM, and MRS plate medium were used. To evaluate amylase and cellulase, 2% of soluble starch (SS) and carboxymethyl cellulose (CMC) were added, and a mixture of iodine and potassium iodine was used as the test solution. Protease and xylanase were evaluated by adding 2% of skim milk and birchwood xylan, respectively, and tested using Congo Red solution. Lipase was prepared by adding 0.3% tween 80 (Sigma-Aldrich Corp., St. Louis, MO, USA) and 1.5% glucose tributyrate (Sigma-Aldrich Corp., St. Louis, MO, USA) to spirit blue media, and cultured overnight to measure transparent rings. Enzymatic activities were compared between 24 h and 48 h samples.

2.8. Statistical Analysis

This study used the Statistical Package for Social Science (SPSS, IBM, Version 24) for statistical analysis. For multiple comparisons between means by treatment group, analysis of variance (ANOVA) was performed using a generalized linear model (GLM), and one-way ANOVA and two-way ANOVA were performed using the Tukey’s honestly significant difference (HSD) post hoc test. Significant differences were assessed at the 95% confidence level, and data are expressed as mean values ± standard deviation (SD) or standard errors of means (SEM).

3. Results

3.1. Microbial Population and pH Changes of Fermented Pine Needles

Figure 1 shows the number of viable cells and changes in pH according to the fermentation of each pine needle culture medium. The viable cell count of CON naturally fermented for 48 h was 10.7 (log10 CFU/mL) in LB, 9.0 in YM, and 3.8 in MRS. Both LPF and SPF cultures showed an initial viable cell count of 5.7, and showed an increasing trend until 48 h (LPF: 11.0; SPF: 11.1). The mixed fermentation culture medium, CCF, also increased the number of L. plantarum and S. cerevisiae over time. The pH of all cultures decreased over time, and in the 48 h sample, LPF (pH: 3.9), CCF (4.6), SCF (5.1), and CON (5.5) were lower in the same order.

3.2. Changes in the Bioactivities of Pine Needles following the Fermentation

3.2.1. Antibacterial Activity

Table 2 shows the antibacterial activity results of each pine needle culture over time. CON showed antibacterial activity against one out of ten pathogens, LPF inhibited seven pathogens, SCF two pathogens, and CCF four pathogens. As time passed, CON and SCF showed no difference in antibacterial activity, but LPF and CCF showed increasing antibacterial activity (p < 0.05).

3.2.2. Total Polyphenols and Flavonoids

The TPC and TFC of fermented pine needles over time are shown in Table 3. In the case of TPC, LPF and SCF showed the highest values at 48 h, and in the case of TFC, CON, LPF, and SCF showed the highest contents when fermented for 12 h (p < 0.05). In particular, SCF showed higher levels of TPC and TFC than other fermentation broths (p < 0.05).

3.2.3. Antioxidant Activity

Changes in DPPH and ABTS radical scavenging activity according to fermenting microorganisms and the fermentation time of pine needle culture medium are shown in Figure 2 and Figure 3, respectively. CON maintained a similar DPPH radical scavenging ability regardless of the fermentation time, but LPF, SCF, and CCF gradually increased and showed higher activity compared to the CON (p < 0.05).
In the case of ABTS, the radical scavenging ability of the SCF culture fermented for 48 h was the highest, and the LPF for 48 h showed the lowest activity (p < 0.05).

3.2.4. Antioxidant Activity of Porcine Aortic Endothelial Cell

The results of comparing the antioxidant activity of fermented pine needles using PAE cells are shown in Figure 4. When the cells were treated with LPS, the DCFH-DA-stained area was high, indicating that the PAE cells were well oxidized. There was no difference between CON at 0 h and LPS treatment, but CON at 48 h significantly inhibited cell oxidation (p < 0.05). The 48 h fermentation culture of LPF had no effect on inhibiting PAE cell oxidation by LPS, but the CON and SCF at 48 h fermentation showed the highest antioxidant activity (p < 0.05).

3.3. Enzymatic Activities

Table 4 shows the enzymatic activity results of the agar well diffusion method according to the fermentation time of each pine needle fermentation culture. In the case of amylase, SCF showed higher activity than CCF (p < 0.05), but there was no effect according to fermentation time. Cellulase showed the highest activity at 48 h of CON and 24 h of CCF (p < 0.05), but there was no effect of fermentation time. Protease activity showed similar activity depending on the fermentation broth of pine needles, but the activity increased as the fermentation time increased (p < 0.05). Lipase only showed activity in CON, and the activity was strong as time passed (p < 0.05).

4. Discussion

This study aimed to develop a phytogenic fermented feed additive by applying fermentation microorganisms to pine needles of pine (P. densiflora) widely distributed in Korea. As fermentation microorganisms, L. plantarum and S. cerevisiae, which are native to various natural products and are used in high proportions worldwide, were used alone or in combination. Each fermented pine needle was investigated for its fermentation characteristics and changes in physiological activity, and based on the results, the feasibility of using it as a livestock feed additive was evaluated.
As a result of fermentation, the number of viable cells increased over time in all fermented broths, and the highest number of viable cells was shown at 48 h. pH was also gradually decreased in all cultures until 48 h, and the LPF culture showed the lowest value. Many studies have also reported that the number of viable cells increased with time during fermentation when natural products were fermented with L. plantarum and S. cerevisiae [25,34,35]. In particular, fermentation of L. plantarum gradually decreased the pH, finally reaching the range of 3.5–4.0 [25,36]. This is considered to be due to lactic acid produced by L. plantarum, a LAB, using sugars (fructose, glucose, and sucrose) in natural plants during fermentation [37]. The pH value of CCF, which is higher than LPF and lower than SCF, is also judged to be due to the above reason. On the other hand, the decrease in the pH of SCF is shown to be due to acetic acid generated during S. cerevisiae fermentation [38], and overall, it is judged that fermentation of each pine needle culture medium normally proceeded.
As a feed additive of natural products and useful microorganisms, the evaluation of antibacterial activity against livestock pathogens in functional evaluation is an important indicator and has been widely analyzed [25,39,40]. There are antibacterial substances such as polyphenols, flavonoids, and sulfur compounds in natural products [41,42], and pine needles have also been reported to have antimicrobial activity against various pathogenic microorganisms [43,44]. In particular, the antibacterial activity of natural plants is improved through fermentation, and lactic acid fermentation by lactic acid bacteria can show stronger antibacterial activity by lactic acid and short-chain organic acids produced by LAB [25]. In this study, CON showed antibacterial activity against one of ten pathogens investigated, but LPF, SCF, and CCF showed antibacterial activity against many pathogens. LPF and CCF showed stronger antibacterial activity when fermented for 48 h than when fermented for 24 h. In particular, LPF inhibited the growth of more types of pathogens than CCF. Kothari et al. [25] reported that when Chinese chive was fermented with LAB, it showed stronger antibacterial activity than before fermentation, and the activity retention time was longer. In addition, as a result of ultra-high-performance liquid chromatography-linear trap quadrupole-orbitrap-tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS/MS) analysis, it was confirmed that flavonols (quercetin, kaempferol, myricetin, rutin, and isorhamnetin) exhibiting antibacterial and antiviral activities were increased by the fermentation of Chinese chive. Pine needles also contain flavonoids (quercetin, kaempferol) and organic compounds (bisbenzene, camphene, borneol, phellandrene, terpene, etc.) with antibacterial properties [29,45]. However, additional research is needed regarding the relationship between changes in bioactive compounds following the fermentation of pine needles and improvements in antibacterial activity.
Antioxidants in natural products play a role in maintaining physical health from high temperatures and oxidative stress in livestock, preventing quality deterioration of livestock products, and improving productivity [39,46,47,48]. In general, the antioxidant capacity of natural products is verified by assays using TPC, TFC, DPPH, ABTS, and various stress-induced cells [40,49,50,51]. In this study, looking at the changes in TPC and TFC by pine needle fermentation, LPF and SCF showed the highest TPC values when fermented for 48 h, and TFC increased in most cultures until 12 h and then decreased thereafter. In particular, among the fermented pine needles, SCF showed the highest TPC and TFC levels. DPPH radical scavenging activity improved with the passage of fermentation time in all fermentation broths, and ABTS radical scavenging activity was highest in SCF. TPC and TFC analysis are items that are indicators of antioxidant activity in natural products, and their content varies depending on whether or not the natural product is fermented and the type of fermentation [25,52,53]. Dulf et al. [54] performed solid-state fermentation of plum pomace with Aspergillus niger, resulting in an increase in TPC of about 20–40%, and when fermenting Cordyceps sinensis, TPC and TFC significantly increased with the lapse in fermentation time [55]. Kuo et al. [56] reported that Chenopodium formosanum fermented with L. plantarum showed more phenolic compounds and improved DPPH and ABTS scavenging ability. Hur et al. [57] revealed that a hydrolysis reaction during fermentation by microorganisms increased the amount of phenolic compounds and flavonoids, and that the structural destruction of plant cell walls liberated and synthesized various antioxidant compounds to improve antioxidant activity. Considering this, it is thought that the increase in TPC content and the improvement in antioxidant capacity of the fermented pine needle is due to the free and synthesized phenolic compounds and flavonoids caused by microorganisms affecting the cell wall structure of pine needles during fermentation.
On the other hand, the antioxidant effect using LPS-treated PAE cells was strong in CON and SCF fermented for 48 h, which was similar to the ABTS result. The exposure of cells to LPS causes severe stress, resulting in cell damage, resulting in an imbalance between ROS production and elimination [58,59]. Plants have antioxidant systems such as ascorbate and glutathione metabolites, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzymes to minimize the effects of ROS damage [58,60]. In addition, microorganisms such as LAB and yeast are known to inhibit peroxidation by increasing the activity of SOD, CAT, and GPx [61,62]. Lee et al. [63] fermented Astragalus membranaceus with L. plantarum and, as a result, upregulated SOD, CAT, and GPx compared to before fermentation, resulting in high antioxidant activity on H2O2-induced HepG2 cells. In this study, it is judged that the fermentation of pine needles significantly prevented oxidation of LPS-induced PAE cells through the interaction between antioxidants contained in pine needles and fermenting microorganisms. However, further research is needed on the investigation of antioxidant systems that are improved through microbial fermentation and the mechanisms that act on oxidative cells.
The ability of useful microorganisms to produce enzymes improves the ability of animals to digest nutrients, and thus improves feed utilization efficiency [64]. In particular, it is known that the production capacity of enzymes is improved by the fermentation of natural products [65]. Each fermented pine needle broth exhibited different enzymatic activity, and the activity increased, decreased, or did not change with fermentation time. Microorganisms, including yeast, produce enzymes such as lipase, amylase, and protease [66]. In addition, microorganisms including Bacillus are known to improve their ability to produce existing enzymes as fermentation proceeds [57]. Xu et al. [67] reported that amylase, carboxymethyl cellulase, lipase, and protease increased over time during the fermentation of various microorganisms. Fermentation of rose (Rosa rugosa thunb) residue by L. plantarum and B. subtilis improved functional enzymatic activities (α-amylase, protease) compared to before fermentation [68]. Among the results of this study, the different enzymatic activity according to pine needle fermentation is judged to be due to the interaction between the number of microorganisms increased by fermentation and pine needles and fermenting microorganisms.

5. Conclusions

In this study, the fermentation characteristics were investigated for the first time using pine needles of P. densiflora, one of the Korean pine trees, and its applicability as a feed additive was confirmed based on antibacterial activity against livestock pathogens, antioxidant activity, and enzymatic activity. Each of the pine needle fermentation broths, CON, LPF, SCF, and CCF, was fermented as the number of microorganisms and pH changed over time. As a result of analyzing the antibacterial activity against the pathogens of livestock, LPF showed the highest antibacterial activity, especially after the fermentation process. TPC showed the highest values in LPF and SCF samples fermented for 48 h, and DPPH improved as all pine needle cultures were fermented, but antioxidant activity using ABTS and PAE cells was highest in SCF. In conclusion, LPF exhibits strong antibacterial activity by fermentation and is judged to be effective in inhibiting the growth of livestock pathogens and preventing diseases, and SCF improves the antioxidant capacity to help mitigate adverse effects caused by stress and maintain livestock product quality. Therefore, it is expected that fermented pine needles can be used as a natural feed additive that has a positive effect on animal husbandry.

Author Contributions

Conceptualization, Y.-A.H. and S.-K.K.; methodology, Y.-A.H., S.-G.H. and S.-K.K.; software, Y.-A.H. and W.-D.L.; validation, Y.-A.H., W.-D.L. and S.-K.K.; formal analysis, Y.-A.H. and W.-D.L.; investigation, Y.-A.H., W.-D.L., J.K., S.K., M.-G.C., J.-Y.O. and S.-W.J.; resources, Y.-A.H. and S.-K.K.; data curation, Y.-A.H., J.K. and W.-D.L.; writing—original draft preparation, Y.-A.H. and W.-D.L.; writing—review and editing, Y.-A.H., W.-D.L., J.K., S.K., M.-G.C., J.-Y.O., S.-W.J., S.-G.H. and S.-K.K.; visualization, Y.-A.H. and W.-D.L.; supervision, S.-K.K.; project administration, S.-K.K.; funding acquisition, S.-K.K. All authors have read and agreed to the published version of the manuscript.

Funding

This paper was supported by Konkuk University Researcher Fund in 2022.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are available on request from the corresponding author.

Acknowledgments

The author W.-D. Lee would like to acknowledge the 2023 RDA Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00415 g001 550
Figure 1. Change in viable cell count and pH according to fermentation time of pine needle culture medium. (A) no probiotic inoculation; Control, (B) L. plantarum SK4315 fermentation, (C) S. cerevisiae SK3587 fermentation, (D) mixed fermentation of L. plantarum SK4315 and S. cerevisiae SK3587.
Figure 1. Change in viable cell count and pH according to fermentation time of pine needle culture medium. (A) no probiotic inoculation; Control, (B) L. plantarum SK4315 fermentation, (C) S. cerevisiae SK3587 fermentation, (D) mixed fermentation of L. plantarum SK4315 and S. cerevisiae SK3587.
Fermentation 09 00415 g001
Fermentation 09 00415 g002 550
Figure 2. Change in DPPH radical scavenging activity according to the fermentation time of pine needle culture medium. (A) No probiotic inoculation; Control, (B) L. plantarum SK4315 fermentation, (C) S. cerevisiae SK3587 fermentation, (D) mixed fermentation of L. plantarum SK4315 and S. cerevisiae SK3587. a–d Means with different superscripts in same column are significantly different (p < 0.05).
Figure 2. Change in DPPH radical scavenging activity according to the fermentation time of pine needle culture medium. (A) No probiotic inoculation; Control, (B) L. plantarum SK4315 fermentation, (C) S. cerevisiae SK3587 fermentation, (D) mixed fermentation of L. plantarum SK4315 and S. cerevisiae SK3587. a–d Means with different superscripts in same column are significantly different (p < 0.05).
Fermentation 09 00415 g002
Fermentation 09 00415 g003 550
Figure 3. Comparison of ABTS radical scavenging ability of each fermented pine needle culture medium. a–d Means with different superscripts in same column are significantly different (p < 0.05).
Figure 3. Comparison of ABTS radical scavenging ability of each fermented pine needle culture medium. a–d Means with different superscripts in same column are significantly different (p < 0.05).
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Fermentation 09 00415 g004 550
Figure 4. Photo and graph of superoxide scavenging activity of porcine aortic endothelial (PAE) cells by pine needle fermentation broth treatment. (A) PAE cell without LPS inoculation, (B) PAE cell with LPS inoculation, (C) 0 h fermented pine needle in LPS-treated cell, (D) 48 h fermented CON in LPS-treated cell, (E) 48 h fermented LPF in LPS-treated cell, (F) 48 h fermented SCF in LPS-treated cell, (G) 48 h fermented CCF in LPS-treated cell, (H) comparison of antioxidant activity of lipopolysaccharide (LPS)-treated PAE cells according to pine needle fermentation broth treatment. a–b Means with different superscripts in same column are significantly different (p < 0.05).
Figure 4. Photo and graph of superoxide scavenging activity of porcine aortic endothelial (PAE) cells by pine needle fermentation broth treatment. (A) PAE cell without LPS inoculation, (B) PAE cell with LPS inoculation, (C) 0 h fermented pine needle in LPS-treated cell, (D) 48 h fermented CON in LPS-treated cell, (E) 48 h fermented LPF in LPS-treated cell, (F) 48 h fermented SCF in LPS-treated cell, (G) 48 h fermented CCF in LPS-treated cell, (H) comparison of antioxidant activity of lipopolysaccharide (LPS)-treated PAE cells according to pine needle fermentation broth treatment. a–b Means with different superscripts in same column are significantly different (p < 0.05).
Fermentation 09 00415 g004
Table
Table 1. The nutrient composition of pine needles in P. densiflora.
Table 1. The nutrient composition of pine needles in P. densiflora.
ItemsAmount
Moisture, %14.2 ± 0.120
Crude protein, %27.0 ± 0.056
Crude fat, %4.56 ± 0.141
Crude fiber, %11.8 ± 0.134
Crude ash, %14.8 ± 0.084
Calcium, %0.715 ± 0.007
Phosphorus, %0.835 ± 0.021
Acid detergent fiber, %15.0 ± 0.176
Neutral detergent fiber, %19.2 ± 0.268
Potassium, ppm46,591 ± 23.2
Magnesium, ppm2092 ± 14.8
Sodium, ppm362 ± 18.6
Iron, ppm1080 ± 11.1
Sulfur, ppm1329 ± 48.4
Values shown are expressed as mean ± standard deviation. Percentages (%) are expressed on a dry basis.
Table
Table 2. Changes in antibacterial activity according to the fermentation time of each pine needle culture medium.
Table 2. Changes in antibacterial activity according to the fermentation time of each pine needle culture medium.
Pathogen BacteriaDiameter of Inbihition Zone (mm)
CON 1LPFSCFCCF
24 h48 hSEM 2p-Value24 h48 hSEMp-Value24 h48 hSEMp-Value24 h48 hSEMp-Value
Listeria monocytogenes SK7288.678.330.2230.51910.00 b13.00 a0.7640.0219.339.670.2240.51910.00 b12.00 a0.516<0.05
Clostridium perfringens SK870ND 3ND--7.009.000.6830.158NDND--6.33 b8.33 a0.494<0.05
Enterotoxigenic E. coli SK871NDND--NDND--NDND--NDND--
Burkholderia contaminans SK875NDND--7.679.000.5580.275NDND--NDND--
Haemopillus
parasuis SK890		NDND--6.338.000.4770.0676.676.330.3420.6786.33 b8.00 a0.401<0.05
Staphylococcus
aureus SK2027		NDND--6.67 b8.00 a0.333<0.05NDND--7.00 b8.00 a0.224<0.05
Haemophilus
somnus SK2047		NDND--12.67 b17.33 a1.065<0.05NDND--9.33 b14.67 a1.265<0.05
Salmonella gallinarum SK3359NDND--NDND--NDND--NDND--
E. coli SK4230NDND--NDND--NDND--NDND--
Pantoea agglomerans SK4295NDND--8.33 b10.67 a0.619<0.05NDND--NDND--
1 CON, no probiotic inoculation; LPF, L. plantarum SK4315 fermentation; SCF, S. cerevisiae SK3587 fermentation; CCF, co-culture fermentation. 2 SEM, standard error of the mean. 3 ND, not detected. a,b Value with different superscripts within a row are significantly different (p < 0.05). The hole size was 6 mm in diameter.
Table
Table 3. Changes in total polyphenol and total flavonoid contents according to fermentation time of each pine needle culture medium.
Table 3. Changes in total polyphenol and total flavonoid contents according to fermentation time of each pine needle culture medium.
Items 1Fermentation Time (h)SEM 2p-Value
048122448
Total polyphenol contents, mg/mL
CON2.64 cC2.19 dC1.96 eD2.87 aB2.79 abB2.71 bcB0.081<0.05
LPF2.85 bcB2.02 dD2.83 cB2.94 abAB2.94 abA3.01 aA0.083<0.05
SCF3.00 aA2.84 cA2.91 bA2.99 abA2.95 abA3.03 aA0.018<0.05
CCF2.52 D2.40 B2.38 C2.45 C2.48 C2.45 C0.018<0.05
SEM0.0580.0940.1170.0660.0580.074
p-value<0.05<0.05<0.05<0.05<0.05<0.05
Total flavonoid contents, mg/mL
CON7.05 bB5.99 cC5.29 dC8.59 aAB8.40 aA7.20 bA0.293<0.05
LPF7.91 bA5.46 cD8.32 aA8.35 aB7.94 bB7.67 bA0.245<0.05
SCF8.36 bA7.96 cA8.32 bA8.89 aA8.11 bcB7.60 dA0.101<0.05
CCF6.67 aB6.45 abB6.24 bB6.64 abC6.50 abC6.25 bB0.057<0.05
SEM0.2170.2820.4010.2680.2220.192
p-value<0.05<0.05<0.05<0.05<0.05<0.05
1 CON, no probiotic inoculation; LPF, L. plantarum SK4315 fermentation; SCF, S. cerevisiae SK3587 fermentation; CCF, co-culture fermentation. 2 SEM, standard error of the mean. a–e Value with different superscripts within a row are significantly different (p < 0.05). A–D Value with different superscripts within a column are significantly different.
Table
Table 4. Changes in digestive enzymatic activity according to the fermentation of pine needle culture medium.
Table 4. Changes in digestive enzymatic activity according to the fermentation of pine needle culture medium.
Items 1Fermentation Time (h)Enzymatic Activity (mm)
AmylaseCellulaseProteaseLipase
CON24ND 29.00 ± 0.008.00 ± 0.008.33 ± 0.58
4810.33 ± 0.589.00 ± 1.0011.33 ± 1.53
LPF24ND8.67 ± 1.158.00 ± 0.00ND
489.67 ± 0.588.33 ± 0.58
SCF2418.00 ± 0.00ND8.67 ± 0.58ND
4818.33 ± 0.589.00 ± 0.00
CCF2412.67 ± 0.5810.00 ± 0.588.33 ± 0.58ND
4812.33 ± 0.588.67 ± 0.589.00 ± 0.00
p-valueProduct<0.05<0.050.163<0.05
Fermentation time1.0000.273<0.05<0.05
Product × Fermentation time0.468<0.050.618<0.05
1 CON, no probiotic inoculation; LPF, L. plantarum SK4315 fermentation; SCF, S. cerevisiae SK3587 fermentation; CCF, co-culture fermentation. 2 ND, not detected. Value represents the mean ± SD of three repeats.
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Hwang, Y.-A.; Lee, W.-D.; Kim, J.; Kim, S.; Choi, M.-G.; On, J.-Y.; Jeon, S.-W.; Han, S.-G.; Kim, S.-K. In Vitro Fermentation Characteristics of Pine Needles (Pinus densiflora) as Feed Additive. Fermentation 2023, 9, 415. https://doi.org/10.3390/fermentation9050415

AMA Style

Hwang Y-A, Lee W-D, Kim J, Kim S, Choi M-G, On J-Y, Jeon S-W, Han S-G, Kim S-K. In Vitro Fermentation Characteristics of Pine Needles (Pinus densiflora) as Feed Additive. Fermentation. 2023; 9(5):415. https://doi.org/10.3390/fermentation9050415

Chicago/Turabian Style

Hwang, Young-A, Woo-Do Lee, Juhyeon Kim, Solhee Kim, Min-Gyung Choi, Jeong-Yeon On, Sang-Woo Jeon, Sung-Gu Han, and Soo-Ki Kim. 2023. "In Vitro Fermentation Characteristics of Pine Needles (Pinus densiflora) as Feed Additive" Fermentation 9, no. 5: 415. https://doi.org/10.3390/fermentation9050415

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