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Article

Effect of Molasses Addition on the Fermentation Quality and Microbial Community during Mixed Microstorage of Seed Pumpkin Peel Residue and Sunflower Stalks

by
Ning Zhang
†,
Yajie Zhou
†,
Adnan Ali
,
Tengyu Wang
,
Xinfeng Wang
* and
Xinwen Sun
*
College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2024, 10(6), 314; https://doi.org/10.3390/fermentation10060314
Submission received: 15 May 2024 / Revised: 4 June 2024 / Accepted: 11 June 2024 / Published: 13 June 2024
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
This study investigated the effect of molasses addition on the fermentation quality, chemical composition, and bacterial community of seed pumpkin peel residue (SPPR) mixed with sunflower straw (SS) in microstorage feed. Molasses additions on a dry matter basis (DM) were divided into three groups: 0% control (CON), 1% (MA), and 2% (MB), and the raw materials underwent mixed microstorage for a period of 60 days. MA exhibited the highest content of dry matter (DM), the lowest content of neutral detergent fiber (NDF), acid detergent fiber (ADF), and ammoniacal nitrogen (NH3-N), as well as the lowest microbial diversity abundance and the highest relative abundance of lactobacilli (p < 0.05). MB demonstrated the highest crude protein (CP) content and acetic acid (AA) and propionic acid (PA) concentrations, with the lowest pH. In conclusion, the addition of molasses could enhance the quality of mixed microsilage feeds composed of seeded pumpkin peel pomace (SPPR) and sunflower straw (SS), with the optimal addition of molasses being 1% on a DM basis.

1. Introduction

Seed pumpkin (Cucurbita pepo) is a general term for pumpkin species whose seeds are utilized as the primary processing object or edible organ, belonging to the annual herbaceous plant category. Seed pumpkin pips refer to the by-products such as pulp and rind remaining after seed extraction. Relevant research [1] shows that seed pumpkin pips contain a crude protein level of 15%, making them suitable as high-quality feed for ruminants. However, due to their high moisture content, they typically undergo gradual decay within about three days under natural conditions and are more challenging to preserve.
Sunflower, belonging to the Asteraceae sunflower genus, comprises annual tall herbaceous plants. While sunflower straw contains a significant amount of fiber, its protein content, sugar content, and energy value are relatively low. Sunflowers are commonly utilized as roughage for ruminants as an alternative to conventional silage [2,3]. In a related study, it was demonstrated that feeding sunflower residue silage to Mohabadinai dairy goats led to a notable increase in serum LDL (low-density lipoprotein). Moreover, it was observed that LDL levels decreased with an increased addition of sunflower residue silage. Furthermore, the study illustrated that incorporating sunflower residue silage into the normal diet could allow for substituting 30% of conventional silage without adversely affecting milk production and composition [4].
Molasses, a viscous, dark brown, semi-fluid object containing mainly sucrose, is a by-product of the beet sugar industry, which contains nutrients such as sugars, minerals, vitamins, proteins, and biologically active substances including phospholipids, oligosaccharides, and phenols [5]. Molasses serves not only as an energy feed to enhance feed conversion efficiency and animal production performance [5,6,7] but also as a silage fermentation promoter, rapidly reducing the pH value of silage. Simultaneously, it effectively inhibits the production of butyric acid and protein hydrolysis, thereby improving silage fermentation quality [8,9]. Molasses is commonly used as an additive in straw microsilage, boasting a high soluble carbohydrate content and containing a small amount of nutrients such as soybean flavonoids, proteins, and flavor-enhancing substances like glutamic acid and aspartic acid. This makes it suitable for application in straw fermentation feed [10,11,12,13]. In a study by W. Jian et al. [14], using a mixture of rice straw, local vegetable by-products, and alfalfa in silage fermentation, concentrations of 2.5% and 5.0% molasses were, respectively, added, demonstrating an improved fermentation quality of mixed silage. Specifically, the addition of 2.5% molasses increased the lactic acid content and reduced the pH. Broderick G.A et al. [15] added 0.5%, 1%, and 2% molasses to cassava silage, observing a decrease in the pH value with an increasing molasses content. Similarly, Ref. [16] introduced lactic acid bacteria to corn silage, noting a decrease in the pH value with increased molasses addition. Additionally, molasses fermentation broth was found effective in reducing the acetic acid content and pH at the onset of fermentation, thereby prolonging the aerobic stability of corn silage.
Hence, through assessing the impact of varying levels of molasses addition on the fermentation outcome of mixed microstorage involving seed pumpkin peelings and sunflower straw, this study aimed to address the challenge of preserving seed pumpkin peelings while enhancing the utilization rate of sunflower straw. Additionally, this study sought to offer a scientific basis for enriching feed sources in the animal husbandry industry.

2. Materials and Methods

2.1. Test Materials

The experiment utilized seed pumpkin residue and sunflower straw sourced from the 187th Corps., 10th Agricultural Division, Xinjiang Production and Construction Corps. Mature seed pumpkins and sunflower stalks were gathered and processed: seed pumpkins were de-seeded, and sunflower stalks were separated from the disk and subsequently crushed into 1–2 cm sizes. The seed pumpkin residue was chopped into pieces measuring 1–2 cm. The nutritional composition of the raw materials is outlined in Table 1. Crushed seed pumpkin residue and sunflower stalks were combined at a ratio of 1:1 based on dry mass. Three levels of molasses were applied: CON (0% molasses addition), MA (1% molasses addition), and MB (2% molasses addition). Molasses is a thick, dark brown, semi-fluid object that contains mostly sucrose and is a by-product of the sugar industry. Without heating when used, it is evenly mixed with water to make a solution and sprayed into the raw material to be fermented. Each treatment corresponds to 6 replicates. During the fermentation of packets, each treatment group produced 12 packets. After the fermentation, 6 packets were randomly selected for chemical composition, fermentation quality and microbial diversity detection. Additionally, 0.2% of a composite bacterial solution (comprising strains of Lactobacillus, Pseudomonas prionis, Saccharomyces cerevisiae, Pseudomonas tropicalis, Bacillus subtilis, Bacteroides albicans, D-FGP bacteria, and D-CF bacteria), 0.2% urea, and 0.2% crude salt were added. The moisture content was adjusted to 65–70%. A fully automatic baling and wrapping machine (RX-DK5252C, Heze, China) was employed to seal the mixture into 80 kg bales, each with 6 layers of wrapping film, resulting in a total of 18 bales. Subsequently, the bales were subjected to fermentation at room temperature for 60 days. After 60 days in the parcel, consider its horizontal surface to display a cross. Focus on where the four lines meet, for center point sampling. Use a rotary barrel sampler to drill into the sample here, to a depth of the parcel of 25–45 cm, and then pull out the rotary sampler. Take out the material and mix it thoroughly, and use the four-part method to reduce the sample to 400–500 g. Transfer the reduced sample into the sampling bag quickly, exclude the air to form a vacuum, and seal it for preservation.

2.2. Nutrient Analysis

The feed samples were initially dried in an oven (LBAO-250; STIK Instrument Equipment Shanghai Co., Ltd., Shanghai, China) at 65 °C. Following drying, the samples were pulverized using a pulverizer (FS200; Guangzhou Bomin Electrical and Mechanical Equipment Co. Ltd., Guangzhou, China) and passed through a 40-mesh sieve for uniformity. The determination of dry matter (DM), crude protein (CP), crude ash (Ash), and crude fat (EE) was conducted following the method outlined by L. Zhang [17]. Additionally, neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose (HC) were determined according to the method described by Van Soest [18].

2.3. Fermentation Quality Measurement

We weighed 35 g of thoroughly mixed microstorage sample and combined that with 100 mL of distilled water. The mixture was then extracted at 4 °C for 24 h. Subsequently, the extract was filtered through four layers of gauze and qualitative filter paper. Immediately following filtration, the pH value of the extracted solution was determined using a digital pH meter (PH8180-0-00; Smart Sensor Co., Ltd., Dongguan, China). The remaining filtrate was transferred into a centrifuge tube and stored frozen at −20 °C for subsequent analysis. The ammoniacal nitrogen concentration was determined using the phenol–sodium hypochlorite colorimetric method [19]. The lactic acid concentration was determined utilizing a lactic acid kit (Nanjing Jianjian Bioengineering Institute, Nanjing, China). The soluble sugar concentration (WSC) was determined via the anthrone–sulfuric acid colorimetric method. Volatile fatty acids were analyzed using high-performance liquid chromatography (1260 Infinity II; Agilent Technologies, Inc., Waldbronn, Germany), following the method described by [20].

2.4. Microbiological Data Analysis

After weighing 20 g of microstorage feed samples into a triangular bottle, 180 mL of deionized water was added. The mixture was then immersed at 4 °C on shaking beds for 4 h. Following immersion, the microstorage feed samples were filtered through four layers of gauze and quantitative filter paper to obtain the leaching solution. The filtrate was then centrifuged at 10,000 rpm for 15 min. After centrifugation, the supernatant was removed, and the precipitate was dissolved with 1 mL of sterile PBS solution. The dissolved precipitate was collected in a 1 mL centrifuge tube for subsequent extraction of the total DNA from mixed microstorage microorganisms. The remaining samples underwent total microbial DNA extraction using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA), following the instructions provided with the DNA extraction kit. The concentration and quality of DNA were determined by agarose gel electrophoresis. Subsequently, the DNA samples were stored in the refrigerator at −20 °C for future use.
The DNA samples that were detected underwent 16S rRNA sequencing at Lynn Biotechnology (Shanghai, China). The main steps of the sequencing process were as follows: The extracted DNA was used as a template for PCR amplification of the V3-V4 region of the bacterial 16S rRNA. The primers used for amplification were 515F (GTGCCAGCMGCCGCGG) and 907R (CCGTCAATTCMTTTRAGTTT). PCR amplification was conducted under the following conditions: initial denaturation at 95 °C for 2 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The PCR reaction system comprised a 20 μL mixture containing 4 μL of 5× FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL (5 μM) of each primer, 0.4 μL of FastPfu polymerase, and 10 ng of DNA template. Each sample was subjected to three replicates. Amplicons were extracted from a 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA).
Purified PCR products were quantified using the Qubit 3.0 fluorometer (Life Technologies, State of California, CA, USA), and amplicons with distinct barcodes, totaling 24, were pooled in equal proportions. Illumina paired-end libraries were subsequently generated from the pooled DNA samples following the standard Illumina DNA genomic library preparation protocol. The constructed amplicon libraries underwent paired-end sequencing (2 × 250) on the Illumina MiSeq platform (BlOZERON Co., Ltd., Shanghai, China). Raw sequencing reads have been deposited into the NCBI Sequence Read Archive (SRA) database. Genomic libraries were prepared and sequenced using the Illumina PE250 platform(Shanghai BIOZERON Co., Ltd., Shanghai, China), and the resulting data were analyzed using QIIME2 software. The sequence data generated in this study have been archived in the Sequence Read Archive (SRA) under accession number PRJNA1102927.

2.5. Data Processing and Analysis

One-way analysis of variance (ANOVA) was conducted using SPSS 23.0 software to assess differences in chemical composition, fermentation quality, and alpha diversity. Multiple comparisons were performed employing Duncan’s method (SPSS 26.0, Chicago, IL, USA). Results are presented as the mean ± standard error (SD). A significance level of p > 0.05 indicates non-significant differences, p < 0.05 denotes significant differences, and p < 0.01 indicates highly significant differences. Stacked histograms were generated using Origin 2021 (OriginLab Corporation, Northampton, MA, USA), while PCoA plots, Wayne plots, LEfSe analysis, and heat maps were created in 15 March 2024 utilizing the online platform https://cnsknowall.com/#/HomePage.

3. Results

3.1. Chemical Composition

The MA group exhibited the highest dry matter content, significantly surpassing that of the CON group (Table 2) (p < 0.001). Additionally, the NDF and ADF contents of the MA and MB groups were lower compared to that of the CON group (p < 0.05). Moreover, the CP content in the MB group was notably higher than those of the MA and CON groups (p < 0.001). Conversely, the ash content in the CON group was significantly lower than those of the MA and MB groups (p < 0.001). There were no significant differences observed in HC and EE between the groups (p > 0.05).

3.2. Fermentation Quality

In Table 3, it can be observed that both the pH and ammoniacal nitrogen content of the MA and MB groups were significantly lower compared to those of the CON group (p < 0.05), with the MB group exhibiting the lowest pH. Furthermore, the acetic acid and total volatile fatty acid content of the MB group were significantly higher than those of the CON and MA groups (p < 0.05). Conversely, the propionic acid content of the MA group was significantly lower compared to those of the CON and MB groups (p < 0.05). However, the contents of soluble carbohydrates and lactic acid did not exhibit significant differences among the groups (p > 0.05). Additionally, butyric acid was not detected in any treatment groups.

3.3. Microbial Communities

According to the OTU clustering analysis depicted in Figure 1a, the CON group exhibited 215 unique OTUs, while the MA and MB groups had 105 and 412 unique OTUs, respectively, resulting in a total of 835 OTUs across all three groups. The coverage rate of the samples in each group exceeded 99%, as shown in Table 4, indicating their representativeness. The Shannon index of the MA group was significantly lower than that of the CON group (p < 0.05), while no significant differences were observed among the MB, CON, and MA groups (p > 0.05). Conversely, the Simpson index of the MA group was significantly higher compared to that of the CON and MB groups (p < 0.05), with no significant difference observed between the CON and MB groups (p > 0.05). There were no significant differences in the Chao1 and ACE indices between the groups (p > 0.05). PCoA analysis (Figure 1b) revealed a substantial difference in microbial community structure between the MA and CON groups (p-value = 0.001).
At the phylum level (Figure 1c), Phylum Thicket emerged as the dominant phylum, with no significant differences observed in the relative abundances of phyla between the groups (p > 0.05).
At the genus level, Levilactobacillus, Loigolactobacillus, and Pediococcus emerged as the dominant genera (Figure 1d). Levilactobacillus exhibited the highest abundance in the MB group, while Loigolactobacillus and Pediococcus were most abundant in the MA group. Genera with relative abundances greater than 0.01 were analyzed for differences (Table 5).
The relative abundances of Levilactobacillus in the MA and MB groups were significantly higher compared to that in the CON group (p < 0.05). Additionally, the relative abundance of Pediococcus in the MA group was significantly higher than those in the CON and MB groups (p < 0.001). Conversely, the relative abundances of Lactiplantibacillus and Lentilactobacillus were significantly lower in both the CON and MB groups compared to in the MA group (p < 0.001).
Further analysis revealed significant correlations between certain genera and various parameters (Figure 2). Levilactobacillus exhibited highly significant positive correlations with CP, AA, PA, NH3-N, and TVFA (p < 0.01) and a highly significant negative correlation with DM (p < 0.01). Pediococcus showed significant negative correlations with AA, PA, NH3-N, and TVFA and significant positive correlations with DM and pH (p < 0.05). Moreover, Lactiplantibacillus was significantly negatively correlated with NDF and ash (p < 0.05), [Eubacterium]coprostanoligenes group_norank was significantly negatively correlated with ash and positively correlated with PA (p < 0.05), Weissella exhibited a significant negative correlation with ADF, Loigolactobacillus was significantly negatively correlated with pH, and Chloroplast_norank was significantly negatively correlated with PA (p < 0.05).

4. Discussion

4.1. Chemical Composition

Existing studies have demonstrated that microstorage dry matter (DM) loss primarily occurs due to the proliferation of undesirable microorganisms [14]. The addition of molasses has been shown to promote the growth of lactobacilli, leading to a rapid decrease in pH, effectively inhibiting the growth of undesirable microorganisms, reducing their degradation of nutrients, and consequently minimizing DM loss. In this study, as molasses addition increased, the DM contents of the MA and MB groups were significantly higher than that of the CON group, indicating that molasses addition effectively mitigated DM loss in mixed microstorage. Additionally, higher neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents indicated a poorer nutritional quality of roughage. NDF and ADF in mixed microstorage feeds with added molasses were significantly reduced (p < 0.05), suggesting that molasses promotes the decomposition of plant cell walls by microorganisms, with the lowest NDF and ADF observed when molasses was added at a level of 0.1% in this experiment. Furthermore, the crude protein (CP) content of microstorage feed significantly increased after adding molasses, likely due to the inherent CP content in molasses itself. Previous studies [8,14,21] have also shown that the CP contents in alfalfa molasses treatment groups were higher compared to in control groups, consistent with the present experiment’s findings. It could be seen that the addition of molasses increased the CP content of the seeded pumpkin peel pomace and sunflower stem mixed microstorage feeds in this experiment, with the CP content peaking when 2% molasses was added.

4.2. Fermentation Quality

The pH, ammoniacal nitrogen (NH3-N) concentration, and organic acid content serve as pivotal indicators for evaluating the fermentation quality of microsilage feeds [22]. When the pH of the microsilage fell below 4.2, this was generally considered indicative of entering the stabilization period of fermentation, where microbial growth is inhibited. In this experiment, pH values ranged from 4.13 to 4.26, with pH decreasing as molasses addition increased. This decline in pH may be attributed to molasses supplying ample nutrients for microbial growth in mixed microsilage, leading to rapid proliferation of lactic acid bacteria during pre-fermentation. Consequently, the overall pH of the fermentation environment rapidly decreased, inhibiting the growth of harmful microorganisms like molds. The slightly higher pH value of 4.26 in the CON group may be attributed to inadequate fermentation substrate provided solely by the water-soluble carbohydrates (WSCs) of seed pumpkin and sunflower stalks. NH3-N concentration reflects protein decomposition by microorganisms in silage feeds, with lower NH3-N indicating reduced microbial protein decomposition, and therefore, a higher fermentation quality and feeding value of silage feeds [23,24,25]. The significant decrease in NH3-N concentration observed in the molasses treatment group in this experiment indicates that molasses addition effectively reduces the NH3-N concentration in mixed microstorage feed, thereby enhancing the fermentation quality. Organic acids in microstorage feed serve as microbial fermentation by-products, reflecting the efficacy of fermentation. A higher lactic acid content, coupled with lower acetic acid and butyric acid levels, signifies superior fermentation. Conversely, elevated acetic acid and butyric acid levels suggest a poor fermentation efficacy [26]. The study of Li et al. [27] demonstrated that molasses addition improved the fermentation quality of mixed oat alfalfa silage, leading to a reduced pH and ammonia-nitrogen concentration, along with an increase in lactic acid content. Consistent with these findings, our experiment also produced an increase in lactic acid content in mixed microsilage following molasses addition. However, the 2% molasses treatment group exhibited a significantly higher acetic acid content compared to the 0% and 1% treatment groups. This divergence in acetic acid content may be attributed to the initial predominance of homo-lactic acid fermentation during the pre-fermentation phase, characterized by low pH tolerance. Subsequently, as fermentation progressed and pH decreased, hetero-lactic acid bacteria, which are more pH-tolerant, became the dominant lactic acid fermenters. This shift led to the production of both lactic acid and acetic acid, with hetero-lactic acid bacteria also converting lactic acid into acetic acid, thereby increasing the acetic acid content in the later stages of fermentation [28]. Similarly, Shao et al. [29] demonstrated that the initial stage of microsilage fermentation primarily involves homo-lactic acid fermentation, dominated by homo-lactic acid bacteria with weak pH tolerance, while the later stage is characterized by hetero-lactic acid fermentation.

4.3. Microbial Communities

It has been established that when sample coverage exceeds 97%, this indicates sufficiently adequate sample collection. In this experiment, the sample coverage of all three groups surpassed 99%, signifying representative sample collection and detection of the vast majority of microorganisms present in the samples. Consequently, the experimental results accurately reflected the microbial composition of the mixed microsilage feed comprising pumpkin peel pomace and sunflower stalks used for seed [30].
Regarding alpha diversity, a higher Chao 1 index signifies greater species richness, a higher Shannon index indicates increased community diversity, a lower Simpson index indicates higher community diversity, and a higher ACE index suggests a greater number of microorganisms. In our experiment, microbial diversity was observed in the order CON > MB > MA. Compared to the CON group, the MA and MB groups exhibited lower Chao 1, Shannon, and ACE indices, alongside a higher Simpson index. This indicates that molasses addition significantly impacted microorganisms in the mixed microstorage of seeded pumpkin peel pomace and sunflower stalks. The decrease in microorganism count following molasses addition can be attributed to the substantial carbon source provided by molasses, promoting the growth and proliferation of lactobacilli. Consequently, lactobacilli quickly became the dominant strain in the fermentation system, leading to a rapid reduction in pH and inhibition of other microorganisms, consistent with findings from the study by R. Luo [31].
In this experiment, Firmicutes and Proteobacteria were identified as the dominant genera in the seed pumpkin peel residue mixed with sunflower stalks for microstorage fermentation. Jazi et al. [32] observed a significant increase in the percentage of Firmicutes in fermented feeds, while Chen [33] demonstrated that Firmicutes play a crucial role in plant fiber decomposition. In our experiment, although the difference in the percentages of Firmicutes among the three groups was not significant (p > 0.05), the percentage in the CON group closely resembled that of the MB group, suggesting that molasses addition may influence microstorage fermentation. Additionally, the percentage of the anamorphic phylum in the MB group was greater than that in the MA group and CON group, with no significant differences observed between the groups. This indicates that molasses addition may increase the percentage of Proteobacteria in the mixed microsilage feed of seed pumpkin peel pomace and sunflower stalks. Zhao et al. [34] reported that Proteobacteria was highly abundant in soil, and yet it accounted for only a small portion in our experiment, likely due to minimal soil contamination during the collection of seed pumpkin peel residue and sunflower stalks.
Zhang et al. [35] discovered that Lactobacillus brevis PL6-1 exhibits rapid sugar utilization for acid production, thereby expediting the fermentation process and enhancing the sensory quality and fermentation flavor of low-salt radish kimchi. These findings align with the results of our experiment. Specifically, the MB group, characterized by the highest molasses addition, demonstrated the highest percentage of Levilactobacillus, accompanied by an elevated total volatile fatty acid content compared to other groups.
Lactobacillus species, including Levilactobacillus, Loigolactobacillus, Pediococcus, Lactiplantibacillus, Lentilactobacillus, and Latilactobacillus, are known for their ability to rapidly decrease pH [36]. These lactobacilli constitute the dominant flora in the mixed microstorage fermentation system of seed pumpkin peel residue and sunflower stalks. The addition of molasses provides essential nutrients for their growth and reproduction, resulting in a higher percentage of lactic acid bacteria in the molasses-treated group compared to the control group. Consequently, the pH of the molasses-treated group is lower than that of the control group, indicating that molasses addition accelerates the fermentation process of mixed microstorage of seed pumpkin peel residue and sunflower stalks.

5. Conclusions

Adequate addition of molasses can enhance the dry matter (DM) and crude protein (CP) content while reducing the crude fiber content in mixed microstorage of seed pumpkin peel residue and sunflower stalks. Furthermore, molasses addition increases the relative abundance of lactobacilli in the mixed microstorage feed, leading to a higher lactic acid content and improved feed quality. Our findings suggest that the optimal quality of seed pumpkin and sunflower straw microstorage is achieved with a 1% molasses addition.

Author Contributions

Conceptualization, X.W. and X.S.; methodology, X.W. and X.S.; software, N.Z. and Y.Z.; validation, Y.Z. and N.Z.; formal analysis, N.Z. and Y.Z.; investigation, X.W. and X.S.; resources, X.W. and X.S.; data curation, X.W.; writing—original draft preparation, N.Z. and T.W.; writing—review and editing, N.Z., Y.Z. and A.A.; visualization, N.Z.; supervision, X.W.; project administration, X.W. and X.S.; funding acquisition, X.W. and X.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Xinjiang Production and Construction Corps. Key Areas of Technological Research and Development (2023AB008), the 8th Group Key Areas of Technological Research and Development (2023NY03), the Scientific Research Project of Shihezi University (KJTP202316), and Xinjiang Production and Construction Corps. Agricultural Key Projects.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequence data reported in this study were archived in the Sequence Read Archive (SRA) under the accession number PRJNA1102927.

Acknowledgments

We would like to express our sincere thanks to Zuoxing Huang, Shihong Mi, Bao Wang, and Cheng cheng Wang for their contributions to the test site and material collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 10 00314 g001
Figure 1. Comparing bacterial abundance in fermented pumpkin peel pomace and sunflower straw with different molasses additions using Wayne and PCoA plots. Wayne (a) and PCoA (b) plots of relative abundance of bacteria in pumpkin peel pomace versus sunflower straw for fermentation of seeds with different molasses additions and at the gate level (c) and genus level (d). Abbreviations: CON: 0% molasses addition; MA: 1% molasses addition; MB: 2% molasses addition.
Figure 1. Comparing bacterial abundance in fermented pumpkin peel pomace and sunflower straw with different molasses additions using Wayne and PCoA plots. Wayne (a) and PCoA (b) plots of relative abundance of bacteria in pumpkin peel pomace versus sunflower straw for fermentation of seeds with different molasses additions and at the gate level (c) and genus level (d). Abbreviations: CON: 0% molasses addition; MA: 1% molasses addition; MB: 2% molasses addition.
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Figure 2. Correlation of relative abundance of bacteria at the genus level with the chemical composition and fermentation parameters. Note: Columns of different colors indicate different subgroups; p-values are on the far right; “*” indicates p ≤ 0.05 and “**” indicates p ≤ 0.01.
Figure 2. Correlation of relative abundance of bacteria at the genus level with the chemical composition and fermentation parameters. Note: Columns of different colors indicate different subgroups; p-values are on the far right; “*” indicates p ≤ 0.05 and “**” indicates p ≤ 0.01.
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Table 1. Chemical constituents of seed pumpkin peel and sunflower straw.
Table 1. Chemical constituents of seed pumpkin peel and sunflower straw.
Raw Material 1Index
DMCPNDFADFAshEEWSC
Seed pumpkin peel5.6810.3120.9814.4010.240.2132.74
Sunflower stalks90.239.8945.2436.8710.172.106.37
1 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; Ash, crude ash; EE, crude fat; WSC, soluble carbohydrate. In the table, CP, NDF, ADF, Ash, EE, and WSC are in dry matter (%DM).
Table 2. Effects of molasses addition on the chemical composition during the mixed microstorage of seed pumpkin and sunflower stalks.
Table 2. Effects of molasses addition on the chemical composition during the mixed microstorage of seed pumpkin and sunflower stalks.
Item 1CONMAMBp-Value
Dry matter (%)31.81 ± 0.11 b37.74 ± 0.12 a36.63 ± 0.08 a<0.001
Neutral detergent fiber (%DM)52.26 ± 2.92 a50.46 ± 3.98 b50.73 ± 3.66 b<0.001
Acid detergent fiber (%DM)13.53 ± 1.39 a12.02 ± 1.08 b12.77 ± 0.58 b0.043
Hemicellulose (%DM)38.72 ± 4.0738.44 ± 4.1237.97 ± 3.820.791
Crude protein (%DM)6.30 ± 0.06 c6.51 ± 0.01 b7.50 ± 0.27 a<0.001
Ether extract (%DM)2.95 ± 0.502.79 ± 0.293.16 ± 0.270.261
Crude ash (%DM)16.50 ± 0.27 b18.86 ± 0.57 a17.70 ± 0.95 a<0.001
1 CON, 0% molasses addition; MA, 1% molasses addition; MA, 2% molasses addition. a–c Different letters in the peer data indicate significant differences (p < 0.05), and the same letter indicates non-significant differences (p > 0.05).
Table 3. Effects of molasses addition on the fermentation quality during mixed microstorage of seed pumpkin and sunflower stalks.
Table 3. Effects of molasses addition on the fermentation quality during mixed microstorage of seed pumpkin and sunflower stalks.
Item 1CONMAMBp-Value
pH4.26 ± 0.02 b4.18 ± 0.04 a4.13 ± 0.02 a0.012
Ammonia nitrogen (μg·mL−1)2.19 ± 0.07 a1.73 ± 0.08 b1.85 ± 0.04 b0.006
Water soluble carbohydrate (μg·mL−1)34.87 ± 9.3635.19 ± 10.3634.27 ± 7.960.929
Lactic acid (mmol·mL−1)12.63 ± 1.4314.42 ± 1.6114.20 ± 1.440.112
Acetic acid (μg·mL−1)14.08 ± 1.25 b14.23 ± 2.92 b18.14 ± 1.82 a0.006
Propionic acid (μg·mL−1)2.69 ± 0.16 a2.45 ± 0.12 b2.71 ± 0.13 a0.007
Butyric acid (μg·mL−1)NDNDND-
TVFA (μg·mL−1)16.77 ± 1.38 b16.68 ± 3.00 b20.85 ± 1.84 a0.005
1 TVFA, total volatile fatty acids; ND, not detected. con, molasses added at 0%; MA, molasses added at 1%; MA, molasses added at 2%. a,b Peer data with different letters indicate significant differences (p < 0.05), and the same letter indicates non-significant differences (p > 0.05).
Table 4. Effects of adding molasses on alpha diversity of microorganisms in mixed silage of seed pumpkin and sunflower stalks.
Table 4. Effects of adding molasses on alpha diversity of microorganisms in mixed silage of seed pumpkin and sunflower stalks.
Item 1CONMAMBp-Value
Chao 1 Index1233.39 ± 233.64974.06 ± 213.561072.90 ± 304.540.235
Shannon Index3.78 ± 0.38 a2.99 ± 0.51 b3.55 ± 0.71 ab0.026
Simpson Index0.20 ± 0.04 b0.35 ± 0.06 a0.25 ± 0.07 b0.003
ACE Index1300.98 ± 258.70980.39 ± 218.461147.74 ± 310.180.146
Coverage %99.27 ± 0.1899.10 ± 0.2799.02 ± 0.210.177
1 CON, 0% molasses addition; MA, 1% molasses addition; MA, 2% molasses addition. a,b Different letters in the peer data indicate significant differences (p < 0.05), and the same letter indicates non-significant differences (p > 0.05).
Table 5. Composition of microbial community at the genus level.
Table 5. Composition of microbial community at the genus level.
Item 1CONMAMBp-Value
Levilactobacillus0.28 ± 0.10 b0.37 ± 0.16 a0.46 ± 0.10 a0.020
Loigolactobacillus0.27 ± 0.090.34 ± 0.240.23 ± 0.070.445
Pediococcus0.11 ± 0.02 b0.41 ± 0.29 a0.16 ± 0.03 b<0.001
Lactiplantibacillus0.08 ± 0.02 a0.01 ± 0.01 b0.03 ± 0.01 a<0.001
Lentilactobacillus0.03 ± 0.01 a0.01 ± 0.01 b0.02 ± 0.01 a<0.001
Latilactobacillus0.04 ± 0.030.01 ± 0.020.02 ± 0.010.202
1 CON, 0% molasses addition; MA, 1% molasses addition; MA, 2% molasses addition. a,b Different letters in the peer data indicate significant differences (p < 0.05), and the same letter indicates non-significant differences (p > 0.05).
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MDPI and ACS Style

Zhang, N.; Zhou, Y.; Ali, A.; Wang, T.; Wang, X.; Sun, X. Effect of Molasses Addition on the Fermentation Quality and Microbial Community during Mixed Microstorage of Seed Pumpkin Peel Residue and Sunflower Stalks. Fermentation 2024, 10, 314. https://doi.org/10.3390/fermentation10060314

AMA Style

Zhang N, Zhou Y, Ali A, Wang T, Wang X, Sun X. Effect of Molasses Addition on the Fermentation Quality and Microbial Community during Mixed Microstorage of Seed Pumpkin Peel Residue and Sunflower Stalks. Fermentation. 2024; 10(6):314. https://doi.org/10.3390/fermentation10060314

Chicago/Turabian Style

Zhang, Ning, Yajie Zhou, Adnan Ali, Tengyu Wang, Xinfeng Wang, and Xinwen Sun. 2024. "Effect of Molasses Addition on the Fermentation Quality and Microbial Community during Mixed Microstorage of Seed Pumpkin Peel Residue and Sunflower Stalks" Fermentation 10, no. 6: 314. https://doi.org/10.3390/fermentation10060314

APA Style

Zhang, N., Zhou, Y., Ali, A., Wang, T., Wang, X., & Sun, X. (2024). Effect of Molasses Addition on the Fermentation Quality and Microbial Community during Mixed Microstorage of Seed Pumpkin Peel Residue and Sunflower Stalks. Fermentation, 10(6), 314. https://doi.org/10.3390/fermentation10060314

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