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Article

Evaluation of the Effect of Incorporating Olive Mill Wastewater on Nutrients, Quality, and Bacterial Flora in Fermented Total Mixed Ration

by
Junliang Zhao
1,
Masanori Kagami
1,
Kiminobu Yano
2 and
Kiyonori Kawasaki
1,*
1
Graduate School of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
2
University Farm, Faculty of Agriculture, Kagawa University, Showa 300-2, Sanuki, Kagawa 769-2304, Japan
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(7), 665; https://doi.org/10.3390/fermentation9070665
Submission received: 6 June 2023 / Revised: 13 July 2023 / Accepted: 13 July 2023 / Published: 15 July 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
Olive mill wastewater (OMW), a major by-product of the olive oil production industry, is rich in polyphenolic compounds that impart health benefits to several animals. Here, we evaluated the effects of OMW addition on the nutrients, quality, and bacterial composition of fermented total mixed ration (TMR). Different amounts of OMW (0%, 5%, 10%, and 20% in fresh matter) as a substitute for water were mixed with the formulated TMR and fermented for 4 weeks. At the initial stages of fermentation, OMW significantly altered the dry matter, pH, and lactic acid content; however, it had minor effects on most macronutrients, quality parameters, and bacterial flora in the final TMR product. OMW (10%) supplementation improved the polyphenolic compound content in the fermented TMR. However, OMW (10% and 20%) increased lipid levels beyond the recommended level of 3% in cow diets. OMW supplementation did not affect the microbial composition of TMR, except for an increased abundance of Weissella. These findings suggest that supplementation of 5–10% OMW improves the quality parameters of TMR without affecting the nutrient composition of fermented TMR. OMW can be used in cow feed; however, further studies involving feeding trials are needed to validate the effects of OMW on cows.

1. Introduction

Food manufacturing generates exorbitant waste worldwide, estimated at 2.9 billion tons annually [1]. The sustainable utilization of these agro-industry by-products is a growing concern. In addition, the suboptimal food availability leading to increasing food–feed competition requires increased resource use efficiency. Many agro-industrial by-products are plant stems and oil residues and can be repurposed in feed processing. Several studies have explored the potential of these by-products as ingredients in animal feed. For example, straw and bran, the major by-products of rice, have long been used as livestock feedstuffs. Recently, dried distiller grains with solubles (DDGSs), which are produced during bioethanol production, have been used as feed ingredients [2], while some waste produced during edible oil processing is being extensively explored.
Olive oil, extracted from olives, imparts various benefits, including immunomodulatory and anticancer effects [3]. The demand for olive oil has tripled over the last 60 years, owing to the rising popularity of functional foods among consumers [4]. However, the management and sustainable processing of olive mill waste imposes an environmental challenge, due to the high concentration of degradation-resistant phytotoxic compounds [5]. Olive mill waste is produced in two forms: the solid form that contains olive pomace (husk, pulp, and crushed olive stone) after decentering and the liquid form that consists of washing water and vegetation water during milling and malaxation [6]. These wastes are rich in polyphenols that impart several benefits and are considered natural antioxidants [7]. Studies have shown that only 2% of the polyphenols in olive fruit are found in virgin olive oil, whereas ~45% of the polyphenols are found in olive pomace, and ~53% are present in liquid waste residues [8]. Given the benefits of polyphenolic compounds in the OMW, their pertinency to soil conditioners and fertilizers [9], food rheological additives [10], and cosmetic ingredients [11] has been extensively explored using various physicochemical techniques, such as electrolysis and membrane filtration [12]. However, current technical approaches are limited by the time and costs required for processing and the land availability for managing the accumulated by-products.
The liquid waste from olive oil extraction, also referred to as olive mill wastewater (OMW), is a black acidic emulsion of vegetation water, fruit tissues (mucilage, pectin), and water added during processing (centrifugation and cleaning) [13]. The organic content in OMW typically ranges from 4% to 18% and has a low nitrogen content [5]. The organic matter in OMW is an attractive resource for producing bioenergy and biofuels while lowering greenhouse gas emissions. Additionally, besides the valuable polyphenolic compounds found in OMW, it also contains various organic acids, such as malic and citric acid, as well as sugar polymers, lipids, and minerals. The organic acids in OMW are thought to enhance the quality of the silage [14], while the nutrients, including sugars, lipids, and minerals, have been identified as suitable substrates for microbial production of biopolymers, such as lipases, pectinases, and extracellular polysaccharides [15].
Incorporating olive by-products into livestock feed is a feasible solution for sustainably handling waste while contributing to livestock farming. Research findings have suggested that feeding ruminants a diet containing olive pomace significantly increases the unsaturated fatty acid content in their milk and meat [16,17], which is attributed to the presence of polyphenols. Similarly, the dietary inclusion of OMW-containing polyphenolic compounds enhances the scavenging activity of radicals in livestock and protects against lipid oxidation in meat [18]. OMW has been used as a feed ingredient in animal diets, including formulated feed for poultry and fish, as well as silage for goats.
Total mixed ration (TMR) is a mixture of grass, silage, concentrate, and water to meet the nutritional demands of dairy cows. TMR serves to minimize the sorting behavior of cows by diluting and masking the unpalatable flavor of ingredients, thus providing balanced nutrients. TMR feeding has become the most adopted method nowadays for feeding dairy cows to achieve maximum performance [19]. Valorization of valuable agro-industrial by-products by formulating them into TMR can simultaneously contribute to livestock farming and sustainable environmental development. Nonetheless, no study has evaluated the feasibility of using OMW in fermented TMR.
This study aimed to explore the potential of using OMW as a sustainable feed alternative by evaluating the effect of OMW supplementation on the nutrients, quality, and microbiota composition of fermented TMR. The findings will assist in effectively utilizing OMW and preventing environmental pollution.

2. Materials and Methods

2.1. Preparation of Fermented TMR

One-hundred grams of TMR with different concentrations of OMW (Table 1) was added to a vacuum pack (Hiryu; AsahiKASEI, Tokyo, Japan). After removing the air from the pack using a vacuum sealer (HIPPO; Asahi Industry Co., Ltd., Aichi, Japan), the vacuum-packed TMR was subjected to anaerobic fermentation at 25 °C for 4 weeks. Six TMR replicates were prepared for each group (Control (Ctrl, 0% OMW), OMW5 (5% OMW), OMW10 (10% OMW), and OMW20 (20% OMW)) for measurement of pH, lactic acid concentration, and short-chain fatty acid (SCFA) concentration; ten TMR replicates were prepared for each group for the microbiota assay. The fresh OMW used in this study was obtained from a company (Agri Olive Shodoshima Co. Ltd., Shodo Island, Kagawa, Japan). Olive pomace that was made from olive oil production was separated to feed and OMW by using a decanter-type centrifugal separator at the company.

2.2. Analysis of Proximate Composition

The proximate compositions of OMW and TMR were analyzed according to the AOAC method [20]. The used AOAC methods included AOAC 930.15 for moisture content, AOAC 942.05 for crude ash (CA) content, AOAC 984.13 for crude protein (CP) content, AOAC 920.39C for crude fat (EE) content, AOAC 962.09 for crude fiber (CF) content, and AOAC 973.18 for acid detergent fiber (ADF) content assessments. Neutral detergent fiber (NDF) was analyzed following the procedure described in a previous study [21].

2.3. Measurement of pH

The pH of the fermented TMR was monitored weekly. Twenty grams of the opened fermented TMR was weighed and placed into a 250 mL centrifuge tube along with 180 mL of ultrapure water. The mixture was then allowed to stand for 24 h at 4 °C. Subsequently, the resulting solution was shaken vigorously and filtered using a 5A filter paper (Advantec, Tokyo, Japan) to obtain the extract. The pH of the extracts was measured using a pH meter (HM-30R; DKK-TOA Corporation, Tokyo, Japan).

2.4. Measurement of Lactic Acid and SCFA

The extract of TMR was obtained as described in Section 2.3. For the lactic acid concentration assay, the extract was stored at −20 °C prior to analysis. Lactic acid was quantified weekly using a lactic acid measuring kit (Dojin Chemical, Kumamoto, Japan) according to the manufacturer’s instructions. For the SCFA concentration assay, the extract was immediately processed and derivatized with isobutyl chloroformate according to the method described by Furuhashi et al. [22]. The SCFA content in the TMR was analyzed after four-week fermentation using gas chromatography–mass spectrometry (GC–MS) carried out on a QP2010 SE system (Shimadzu, Kyoto, Japan) following the conditions described by Kawasaki et al. [23].

2.5. Estimation of Volatile Basic Nitrogen (VBN) and V-SCORE

The VBN content in the fermented TMR was measured using the steam distillation method. Briefly, 10 mL of the extract obtained as described in Section 2.3 was placed in distillation equipment (VELP Scientifica, Usmate Velate, Italy); 20 mL of 30% sodium hydroxide was added, and distillation was performed for 4 min. The distilled solution was recovered in a boric acid indicator containing bromocresol green-methyl red at pH 4.65 and then titrated with 0.025 mol/L sulfuric acid solution for VBN concentration measurement. The V-SCORE, which is used for quality evaluation of silage, was reported according to the scoring evaluation table (Table S1), corresponding to the VBN ratio to total nitrogen and SCFA content [24].

2.6. Measurement of Polyphenol Concentration

Polyphenols in the fermented TMR, including oleuropein, hydroxytyrosol, and tyrosol, were determined using reverse-phase high-performance liquid chromatography (HPLC) after pretreatment with OMW and TMR according to the method described by Hattori et al. [25]. Specifically, 1 g of OMW or TMR was weighed and mixed with 25 mL acetonitrile and 25 mL distilled water. The resulting mixture was sonicated for 60 min, and methanol was added to bring the volume to 100 mL. After sonication, 5 mL of the resulting solution was loaded onto a C18 solid-phase extraction column (InertSep Slim-J C18; GL Sciences Inc., Tokyo, Japan) and eluted with 10 mL acetonitrile: distilled water: methanol (1:1:2) solution. The eluted sample was then adjusted to a volume of 20 mL with acetonitrile: distilled water: methanol (1:1:2) solution and filtered using a syringe filter PTFE (0.45 μm/φ13 mm, ASONE, Tokyo, Japan). The obtained sample solution was finally subjected to an HPLC system (Prominence UFLC, Shimadzu, Kyoto, Japan) equipped with an InertSustain C18 column (φ4.6 × 250 mm × 5 µm, GL Sciences Inc., Tokyo, Japan) for polyphenol analysis. The mobile phase comprised acetonitrile, distilled water, and 0.5 mol/L phosphoric acid in a ratio of 210:790:1. The measurement wavelength was set at 280 nm, while the column temperature was maintained at 35 °C. The flow rate was 0.5 mL/min, and the injection volume was 5 µL. Different concentrations of oleuropein (TCI, Tokyo, Japan), 3-hydroxytyrosol (TCI), and 2-(4-hydroxyphenyl) ethanol (TCI) were used to prepare calibration curves to quantify the polyphenol content in the sample solutions.

2.7. Microbiota Analysis in the Fermented TMR

Bacterial DNA was extracted from the opened fermented TMR using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The V3–V4 region of the 16S rRNA was amplified and sequenced using MiSeq (Illumina, Inc., San Diego, CA, USA) following the Illumina protocol. After sequencing, the data were subjected to quality filtering, processing, and analysis using QIIME2 [26]. The SILVA database was used to create a phylogenetic tree.

2.8. Lactic Acid Bacteria Colony Count

The number of lactic acid bacteria was estimated using the poured-plate method. The extract obtained as in Section 2.3 was serially diluted. The diluted extracts were mixed with bromocresol purple (BCP) lactose agar medium and incubated at 37 °C for 48 h. The visible colonies on the surface and within the medium were counted after incubation. Each experiment was performed in triplicates.

2.9. Statistical Analysis

Statistical analyses of proximal and chemical composition data were performed using JASP 0.16.1 (JASP Team, 2022). Significance was tested using the Kruskal–Wallis test, and data with significant differences were further analyzed using Dunn’s test (Bonferroni correction) for multiple comparisons. Statistical significance was set at p < 0.05. The effects of fermentation time and OMW supplementation on dry matter (DM), pH, and lactic acid concentration of the TMR were analyzed using aligned rank transform (ART) ANOVA with the ARTool package [27] in RStudio. The diversity of the microbiota was analyzed using QIIME2 [26]. The Kruskal–Wallis test and permutational multivariate analysis of variance (PERMANOVA) were performed for statistical analysis of the α-diversity (Chao1 and Shannon indices) and β-diversity (unweighted and weighted UniFrac distances), respectively. The α- and β-diversity plots were generated using the phyloseq package [28]. The abundance of each bacterial genus in the microbiota was analyzed using Welch’s t-test in the statistical analysis of metagenomic profiles (STAMP) software [29].

3. Results

3.1. Chemical Composition of OMW

The chemical compositions, including proximate nutrients and polyphenolic compounds in the OMW, are listed in Table 2. The OMW contained 13.07% dry matter, of which EE (23.28%) and CA (14.70%) were dominant. Additionally, the OMW included 10.01% NDF, 9.80% CP, and low proportions of ADF, at 4.46%, and CF, at 2.90%. Furthermore, oleuropein was the most abundant polyphenol detected in the fresh OMW, with a value of 0.79 mg/mL, while the concentrations of hydroxytyrosol and tyrosol were 0.17 mg/mL and 0.09 mg/mL, respectively.

3.2. Dry Matter, pH, and Lactic Acid Concentration Changes in the Fermented TMR

The dynamic changes in DM, pH, and lactic acid concentrations in the fermented TMR are shown in Figure 1. No pattern of change in DM content was observed during fermentation, and the DM content of fermented TMR with different treatments showed irregular fluctuations. Nevertheless, the DM content in the OMW-treated groups was consistently lower than that in the control group throughout the fermentation period (Figure 1A). The pH in the control and OMW groups showed a decreasing trend with increasing fermentation duration. A notable decrease in pH was observed during the initial 7-day fermentation, followed by a slight decrease until the completion of fermentation. In addition, the pH in the OMW groups was higher than that in the control group (Figure 1B). The lactic acid concentration in OMW-treated TMR was lower than in non-treated TMR and varied roughly from 200 mmol/L to 300 mmol/L in the final fermented product (Figure 1C). The effects of fermentation time and OMW supplementation on DM, pH, and lactic acid concentrations are shown in Table 3. Both fermentation time and OMW supplementation significantly affected the DM, pH, and lactic acid concentration in the TMR during fermentation. In contrast, only the interaction between fermentation time and OMW supplementation was significant for pH.

3.3. Nutritional Compositions and Quality Parameters of the TMR after Four-Week Fermentation

The nutritional composition (including macronutrients), VFA concentrations, polyphenol concentrations, and quality parameters (e.g., pH, Lactobacillus colony counts, lactic acid concentration, and V-Score), of the fermented TMR after a four-week fermentation are shown in Table 4. The macronutrients regarding DM, CA, CP, CF, ADF, and NDF were comparable among the different treatments, while the EE values in the fermented TMR increased with increasing concentrations of incorporated OMW. Furthermore, the EE content in fermented TMR was significantly higher in the 10% and 20% OMW-incorporated groups compared to the control group (p < 0.05). Similarly, the pH values of the OMW10 and OMW20 groups were significantly higher than that of the control (p < 0.05). However, no significant differences were observed in the number of viable lactobacilli, organic acids, or polyphenols. Notably, all polyphenols showed the highest values at OMW10 and were not proportional to the amount of OMW.

3.4. Microbiota Composition in Fermented TMR after Four-Week Fermentation

A total of 1,190,360 sequences were obtained from the fermented TMR using the MiSeq sequencing system (Illumina, Inc., San Diego, CA, USA), resulting in the identification of 422 operational taxonomic units through clustering the generated sequences. The diversity of microbiota in the fermented TMR is depicted in Figure 2, showing that there was no significant difference found for either the α-diversity indices (Chao 1: richness, Shannon: evenness) or the β-diversity indices (unweighted UniFrac distance: qualitative, unweighted UniFrac distance: quantitative) between experimental groups. Additionally, the addition of OMW had a minor effect on the distribution of microbial communities in the fermented TMR (Figure S1). The microbiota was dominated by Lactobacillaceae (Table S2), with only Weissella showing significant differences in the abundance of bacterial genera (Figure 3).

4. Discussion

Growing concerns regarding environmental pollution caused by agro-industry by-products have raised increasing interest in the exploitation and recovery of organic waste, including OMW. Over 30 million tons of OMW are generated annually in the Mediterranean basin, in which the global olive oil supply is mainly manufactured and supplied [30]. Unfortunately, a standardized and efficient way of handling OMW is yet to be discovered. Moreover, the seasonal pooling of OMW yields further complicates its management. In this study, to effectively utilize OMW, we evaluated the feasibility of OMW as a feed ingredient in fermented TMR. By repurposing OMW in the fermented TMR, it is possible to compensate for the drawbacks associated with conventional handling approaches, such as bioremediation, physicochemical treatment, and disposal. These approaches are generally constrained by the available land for managing raw OMW, the high costs consumed for setting up the equipment, and the environmental regulations for preventing pollution from hazardous substances [5]. In addition, since fermentation is a less complex and affordable technology, the addition of OMW to fermented TMR is a viable approach that not only allows microbial degradation of the abundant polyphenolic compounds in OMW but also enables the recycling of valuable nutrients present in OMW. To the best of our knowledge, this study was the first to investigate the effect of OMW supplementation on the quality and composition of fermented TMR, which is a typical feed for aiding dairy cow farming profitability. Although animal feeding trials were not conducted in the present investigation, the results, in terms of nutrition and quality parameters of the fermented TMR, suggested that OMW can potentially substitute the water used in TMR mixing and fermentation processes.
The OMW used in this study had a higher DM content (13.07%) and EE levels than those reported in previous studies [31,32,33], which could be due to the high residual levels of olive pomace in the OMW in our study. We showed that oleuropein and tyrosol are the key polyphenolic compounds in OMW. They are commonly characterized as natural antioxidants and have been shown to contribute to host redox status [34,35]. Given the chemical composition of OMW, we speculated that incorporating an appropriate proportion of OMW into the feed could be a feasible way to use and valorize OMW effectively.
In this study, the addition of OMW significantly affected the DM content, pH, and lactic acid concentration of the TMR during the 4 weeks of fermentation. The alteration in initial pH of the OMW-treated TMR could be attributed to acidic compounds, such as phenolic acids [36], which, in turn, could have altered the proliferation and metabolism of microorganisms during fermentation [37], resulting in changes in DM content, pH, and lactic acid concentration of the fermented TMR. However, at the end of fermentation, no significant differences in DM or lactic acid concentrations were observed among the groups. These findings suggest that, although incorporating OMW during fermentation can affect the fermentation process of the TMR, it may have a minor effect on the final product as fermentation progresses.
Nutritional and chemical parameters of fermented TMR are critical indicators for quality monitoring. In this study, the inclusion of OMW did not affect the nutritional parameters of the fermented TMR, such as DM, CP, CA, CF, ADF, and NDF contents, or the chemical parameters, such as Lactobacillus counts, organic acids, and V-score. However, the pH of fermented TMR gradually increased with increasing concentration of incorporated OMW, which may be associated with the bactericidal effects of polyphenolic compounds present in OMW. The high level of addition of acidic phenols and oleuropein in fermented TMR may act synergistically with alcohol from microbial fermentation on peptidoglycan and lipids of the cell envelope, resulting in Gram-negative microbial cell death and acid production decrease [38]. The observed decrease in lactic acid production during fermentation and the decrease in oleuropein production after fermentation in the OMW20 group may collectively provide evidence for this speculation. In addition, with the addition of 10% and 20% OMW, the lipid levels of TMR exceeded the recommended lipid content for cow diets by 3% [39], whereas the OMW5 group was the closest to the control group in all characterized parameters and showed a higher V-score than the control group. Based on the macronutrients and V-score, we speculated that adding 5% OMW to TMR could be used to prepare fermented TMR for dietary cow feed.
Increasing evidence has shown that dietary polyphenols can enhance host performance. For instance, feed supplemented with polyphenolic compounds has been found to reinforce antioxidant defense in lambs [34], rabbits [40], piglets [35], and poultry [41]. Furthermore, polyphenols present in olive by-products did not affect milk yield but improved nutritional traits by increasing unsaturated fatty acids and decreasing atherogenic and thrombogenic indices [42]. In this study, OMW supplementation enriched the fermented TMR with polyphenols. Furthermore, the findings indicated that the addition of 10% OMW to the fermented TMR could be beneficial, as the OMW10 group showed the highest levels of polyphenols among the experimental groups.
Under the present study conditions, no significant differences were observed in the α- and β-diversities of the microbial composition of fermented TMR among the experimental groups. Nevertheless, the addition of OMW significantly reduced the abundance of Weissella in the TMR, which is typically proposed as a probiotic in inoculants [43], and this may have a minor impact on fermented feed quality [44]. In addition, with increased OMW content in TMR, the pH increased, whereas the lactic acid concentration decreased. These results indicate that OMW caused a change in the abundance of bacteria involved in lactic acid production, leading to a difference in lactic acid concentration. A low concentration of lactic acid makes rations more palatable, which, in turn, may contribute to cow performance [45]. However, it did not significantly change the genus-level bacterial abundance in the given blended quantity. Finally, the current study was conducted solely in a controlled laboratory without validation on actual silage production farms and data on the effect of OMW supplementation on the fermented TMR storage, limiting the transferability of the findings to practical applications.

5. Conclusions

The findings in the present study revealed that supplementation of OMW enriched TMR with beneficial polyphenolic compounds without affecting the proximate composition of the TMR. The V-score used to evaluate the quality of fermented TMR indicates that it is possible to completely replace the water added during fermented TMR production with OMW. However, the EE value, pH, lactic acid concentration, and polyphenol content suggest that the amount of OMW should be approximately 5–10% of the fermented TMR. Further studies, such as feeding trials on cows, are necessary to promote the use of OMW in feeds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9070665/s1, Table S1: V-SCORE scoring evaluation in silage on fresh matter; Table S2: The microbial community of different samples in the fermented TMR with different treatments; Figure S1: The principal coordinate plot of the characterized microbial communities in the TMR fermented with different olive mill wastewater (OMW) supplements at the genus level.

Author Contributions

Conceptualization, K.K.; methodology, K.K. and J.Z.; validation, K.K. and J.Z.; formal analysis, K.K., M.K. and J.Z.; resources, K.Y.; data curation, K.K. and J.Z.; draft writing, J.Z.; review and editing, K.K.; visualization, K.K. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

2021 Grant-in-Aid for Budding Research in Kagawa University (R3-1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Sequencing files obtained in FASTQ format were deposited in the NCBI Sequence Read Archive, and the BioProject accession number is PRJNA979910. The raw data used in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00665 g001 550
Figure 1. Dynamic changes in dry matter (A), pH (B), and lactic acid concentration (C) during TMR fermentation with different olive mill wastewater (OMW) supplements. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
Figure 1. Dynamic changes in dry matter (A), pH (B), and lactic acid concentration (C) during TMR fermentation with different olive mill wastewater (OMW) supplements. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
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Fermentation 09 00665 g002 550
Figure 2. Diversity indices of microbial communities in the TMR fermented with different olive mill wastewater (OMW) supplements. (A) α−diversity; (B,C) β−diversity analysis plots of (B) unweighted UniFrac distance and (C) weighted UniFrac distance. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
Figure 2. Diversity indices of microbial communities in the TMR fermented with different olive mill wastewater (OMW) supplements. (A) α−diversity; (B,C) β−diversity analysis plots of (B) unweighted UniFrac distance and (C) weighted UniFrac distance. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
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Fermentation 09 00665 g003 550
Figure 3. Comparative analysis of the microbial communities in the TMR fermented with different olive mill wastewater (OMW) supplements at the genus level. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
Figure 3. Comparative analysis of the microbial communities in the TMR fermented with different olive mill wastewater (OMW) supplements at the genus level. Ctrl: TMR without OMW; OMW5: TMR with 5% OMW; OMW10: TMR with 10% OMW; and TMR with 20% OMW.
Fermentation 09 00665 g003
Table
Table 1. TMR ingredients.
Table 1. TMR ingredients.
IngredientsCtrlOMW5OMW10OMW20
Sorghum silage22.022.022.022.0
Concentrate feed 135.035.035.035.0
Lucerne hay4.54.54.54.5
Timothy hay9.09.09.09.0
Klein grass9.09.09.09.0
Calcium phosphate0.50.50.50.5
Water20.015.010.00.0
Olive mill wastewater0.05.010.020.0
TMR = total mixed ration; OMW = olive mill wastewater. 1 Feed ingredients included grains (corn and soybean) 42%, bran 25%, vegetable oil 14%, other ingredients (alfalfa, zeolite, calcium carbonate, syrup, salt, Bacillus subtilis, and Lactobacillus) 19%.
Table
Table 2. Proximate nutrients and polyphenolic concentration of olive mill wastewater (OMW).
Table 2. Proximate nutrients and polyphenolic concentration of olive mill wastewater (OMW).
Proximate Nutrients (% in Dry Matter)OMW
DM13.07
CA14.70
CP9.80
EE23.28
CF2.90
ADF4.46
NDF10.01
Polyphenolic concentration in the fresh matter (mg/mL)
Oleuropein0.79
Hydroxytyrosol0.17
Tyrosol0.09
DM = day matter; CA = crude ash; CP = crude protein; EE = crude fat; CF = crude fiber; ADF = acid detergent fiber; NDF = neutral detergent fiber.
Table
Table 3. The fermentation time and OMW supplement on dry matter, pH, and lactic acid concentration of TMR.
Table 3. The fermentation time and OMW supplement on dry matter, pH, and lactic acid concentration of TMR.
0 to 4 wkGroupsp-Values of Effects
ItemsCtrlOMW5OMW10OMW20GroupsPeriod (wk)Groups × Period
Dry matter48.33 ± 0.75 a45.31 ± 0.98 bcd43.99 ± 0.71 cd44.49 ± 0.80 d<0.001<0.0010.43
pH4.60 ± 0.10 d4.72 ± 0.09 c4.80 ± 0.07 b4.95 ± 0.07 a<0.001<0.001<0.001
Lactic acid 179.91 ± 17.21 a165.14 ± 15.87 b135.50 ± 11.66 cd132.58 ± 12.39 d<0.001<0.0010.35
Mean values within a row with different superscripts represent the statistical differences in multiple comparisons (p < 0.05).
Table
Table 4. Chemical composition of the TMR after four-week fermentation.
Table 4. Chemical composition of the TMR after four-week fermentation.
ItemCtrlOMW5OMW10OMW20
DM47.07 ± 0.8842.43 ± 0.4644.89 ± 1.5144.68 ± 1.18
CA8.16 ± 0.169.04 ± 0.328.87 ± 0.248.90 ± 0.43
CP17.42 ± 0.5416.67 ± 0.5019.15 ± 0.2917.45 ± 0.90
EE2.14 ± 0.10 cd2.56 ± 0.06 bc3.23 ± 0.22 b3.79 ± 0.12 a
CF14.42 ± 1.7717.76 ± 1.0314.01 ± 1.3416.24 ± 1.55
ADF23.92 ± 2.0221.56 ± 1.9021.04 ± 0.6822.58 ± 2.93
NDF48.48 ± 1.9351.51 ± 1.0956.14 ± 2.7353.19 ± 3.13
pH4.32 ± 0.02 cd4.41 ± 0.01 c4.52 ± 0.02 b4.65 ± 0.05 a
Lactobacilli (Log CFU/g)10.25 ± 0.1910.51 ± 0.0810.35 ± 0.1610.77 ± 0.08
Lactic acid (% of VFA)99.14 ± 0.1098.49 ± 0.6198.25 ± 0.5999.19 ± 0.15
Acetic acid (% of VFA)0.86 ± 0.101.51 ± 0.611.75 ± 0.590.81 ± 0.15
Propionic acid (% of VFA)NDNDNDND
Butyric acid (% of VFA)NDNDNDND
VBN/T-N (%)0.72 ± 0.070.92 ± 0.060.78 ± 0.020.84 ± 0.03
V-SCORE91929092
Oleuropein (mg/kg of DM)NDND315.6 ± 103.2236.4 ± 106.7
Hydroxytyrosol (mg/kg of DM)ND152.6 ± 8.9171.9 ± 12.5166.1 ± 35.3
Tyrosol (mg/kg of DM)NDND15.4 ± 7.312.9 ± 8.7
DM = day matter; CA = crude ash; CP = crude protein; EE = crude fat; CF = crude fiber; ADF = acid detergent fiber; NDF = neutral detergent fiber; VBN = volatile basic nitrogen; T-N = total nitrogen. Mean values within a row with different superscripts represent the statistical differences in multiple comparisons (p < 0.05).
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Zhao, J.; Kagami, M.; Yano, K.; Kawasaki, K. Evaluation of the Effect of Incorporating Olive Mill Wastewater on Nutrients, Quality, and Bacterial Flora in Fermented Total Mixed Ration. Fermentation 2023, 9, 665. https://doi.org/10.3390/fermentation9070665

AMA Style

Zhao J, Kagami M, Yano K, Kawasaki K. Evaluation of the Effect of Incorporating Olive Mill Wastewater on Nutrients, Quality, and Bacterial Flora in Fermented Total Mixed Ration. Fermentation. 2023; 9(7):665. https://doi.org/10.3390/fermentation9070665

Chicago/Turabian Style

Zhao, Junliang, Masanori Kagami, Kiminobu Yano, and Kiyonori Kawasaki. 2023. "Evaluation of the Effect of Incorporating Olive Mill Wastewater on Nutrients, Quality, and Bacterial Flora in Fermented Total Mixed Ration" Fermentation 9, no. 7: 665. https://doi.org/10.3390/fermentation9070665

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