KR102196047B1 - Composition for preservation of a source of livestock feed comprising metabisulfite and preparation method of livestock feed using the same - Google Patents

Abstract

The present invention relates to a composition for preserving feed raw materials containing at least one selected from metabisulfite and organic acids, and to a method for preparing feed using the same, and more specifically, to a fruit and vegetable waste used as feed raw materials. Discards, FVD), a novel use of metabisulfite and organic acids useful for long-term storage, and a silage manufacturing method using the same. As a result of treating at least one selected from metabisulfite and organic acids in fruit and vegetable waste (FVD) according to the present invention, the microbiological stability and freshness of FVD in an aerobic environment were maintained for a longer time, and it was also very effective in preserving nutrients and antioxidant functions. It was confirmed that it was effective. In addition, the silage feed manufacturing method comprising at least one pretreatment step selected from metabisulfite and organic acids in an aerobic environment according to the present invention has the effect of improving the preservation properties during the anaerobic preservation process, and in particular, almond husk and corn gluten together. The co-ensiling of the FVD used showed the best preservation properties.

Description

Composition for preservation of a source of livestock feed comprising metabisulfite and preparation method of livestock feed using the same}

The present invention relates to a composition for preserving feed raw materials containing metabisulfite and a method for preparing feed using the same, and more specifically, useful for long-term storage of fruit and vegetable discards (FVD) used as feed raw materials. It relates to a novel use of metabisulfite and a method for manufacturing silage using the same.

When a nutrient source that cannot be consumed by humans is used as feed for livestock, it can contribute to expanding the food supply by expanding the feed source and alleviating the instability of the feed supply. It is estimated that a third of all processed fruits and vegetables are lost in various parts of the food supply chain (FAO, 2011. The State of Food Insecurity of the World: How Does International Price Volatility Affect Domestic Economies and Food) Security?FAO, WFP, IFAD, Rome, Italy). These substances are potentially nutrient sources and bioactive compounds that are not yet utilized, or self-decompose in landfills and stockpiles, causing major environmental problems.

According to the results of a field investigation conducted by the inventors at a recent commercial packing house, fruit and vegetable wastes (FVDs) are usually deposited in the shed facilities for a week until they move from the source to the recycling center. there was. However, during this period, especially during the hot summer months, FVD tends to decay rapidly due to the high humidity and high content of simple carbohydrates, which accelerate decay and microbial growth. Since such decayed FVD causes environmental problems, it is necessary to immediately treat an effective preservative to the source of FVD in order to prevent or delay the decomposition of FVD by microorganisms.

Sulfiting agent is one of the preservatives widely used in the food industry due to its high cost-effectiveness. Among the sulfite salts, sodium metabisulfite (SMB) is widely used in the feed and food industry, and the silage treated with SMB is effectively preserved due to the oxygen-scavenging activity of SMB, which inhibits the growth of oxygen-dependent microorganisms. Can be. Recently, the inventors have conducted a series of laboratory and pilot-scale conservation experiments confirming the successful storage capacity of SMB. According to this, SMB showed the effect of delaying the deterioration of FVD quality, increasing microbiological stability, and maintaining nutrients during exposure to an aerobic environment. Under this background, the present inventors carried out subsequent studies to further extend the storage stability of SMB-treated FVD in anaerobic conditions and completed the present invention.

Compared to the drying process, which is an energy-intensive and unsustainable method of preservation, ensiling is the long-term preservation of biomass with high moisture content under anaerobic conditions where bacteria produce lactic acid that lowers the pH of the silage to about 4. It can be a simple yet effective way for you. This acidic environment inhibits the growth of harmful parasitic microorganisms during the ensiling fermentation process. However, transport problems such as high transport costs and significant loss of dry matter (DM) during ensiling are pointed out as major problems associated with ensiling of high moisture biomass such as FVD. These issues are of great importance in terms of environmental aspects and economics of the conservation process. In order to cope with these problems, a silage management strategy that can minimize effluent loss and reduce the costs associated with drying and/or transporting high moisture feed ingredients. A method of achieving an appropriate moisture content by adding a DM component has been proposed. However, there have been no reports on the ensiling process of FVDs in which SMBs have been treated and exposed to an aerobic environment.

Republic of Korea Patent Publication No. 10-2016-0058241 International Publication Patent WO2018-081550

An object of the present invention is to provide a composition for preserving feed raw materials comprising metabisulfite and at least one selected from organic acids as an active ingredient.

Another object of the present invention is to provide a method of manufacturing a feed using at least one selected from metabisulfite and organic acids as a raw material for fruit and vegetable waste.

In order to achieve the object of the present invention, the present invention provides a composition for preserving feed raw materials comprising metabisulfite and at least one selected from organic acids as an active ingredient. The feed raw material is preferably fruit and vegetable discards (FVD).

In one embodiment of the present invention, the metabisulfite is sodium metabisulfite (SMB).

In one embodiment of the present invention, the metabisulfite is preferably added in a ratio of 6 to 8 g per 1 kg of fruit and vegetable waste, but is not limited thereto.

In one embodiment of the present invention, the organic acid may be at least one selected from sodium benzoate, potassium sorbate, and sodium nitrite.

In one embodiment of the present invention, the composition may further include almond husk and corn gluten.

In addition, the present invention provides a method for producing a feed using at least one selected from metabisulfite and organic acids. The feed manufacturing method comprises a pretreatment step of preserving after mixing by adding at least one selected from metabisulfite and organic acids to fruit and vegetable waste in an aerobic environment, and then preserving feed based on fruit and vegetable waste. It is a manufacturing method of. In one embodiment of the present invention, the metabisulfite salt used in the pretreatment step may be sodium metabisulfite (SMB). In addition, the organic acid may be at least one selected from sodium benzoate, potassium sorbate, and sodium nitrite.

In one embodiment of the present invention, the metabisulfite is preferably added in a ratio of 4 to 8 g per 1 kg of fruit and vegetable waste, but is not limited thereto.

In one embodiment of the present invention, the metabisulfite is added in a ratio of 6 to 8 g per 1 kg of fruit and vegetable waste, and the preservation may be performed for 6 to 9 days, but is not limited thereto.

In one embodiment of the present invention, the feed is silage. Silage generally refers to a succulent feed obtained by lactic acid fermentation by vacuum storage of feed crops with a high moisture content in a storage container called a silo.

In one embodiment of the present invention, the method of manufacturing the silage feed may include an ensiling step of storing the fruit and vegetable wastes subjected to the pretreatment step in a silo container and sealing it. .

In an embodiment of the present invention, the ensiling step may be performed at room temperature of 20 to 25° C. for 7 to 90 days, but is not limited thereto.

In one embodiment of the present invention, the method of manufacturing the silage feed may be a method of co-ensiling almond husks and corn gluten together with fruit and vegetable wastes subjected to the pretreatment step. have. That is, the method of manufacturing the silage feed may include mixing almond husks and corn gluten together with fruit and vegetable wastes subjected to the pretreatment step, putting them in a silo container, sealing them, and storing them.

In one embodiment of the present invention, the metabisulfite used in the pretreatment step of the silage feed manufacturing method may be sodium metabisulfite (SMB), in which case the sodium metabisulfite is 1 kg of biomass. It is preferable to be contained in the silo container at a rate of 2.4 to 4 g per sugar, but is not limited thereto.

In one embodiment of the present invention, the silage feed manufacturing method is mixed by adding at least one selected from metabisulfite and organic acids to fruit and vegetable waste in an aerobic environment at a rate of 6 g per 1 kg of fruit and vegetable waste. Pre-treatment step of preserving for 7 days after making; Pulverizing the fruit and vegetable wastes passed through the pretreatment step; Mixing almond husk and corn gluten with the pulverized product, wherein the pulverized product is mixed in a ratio of 39.8 to 66.6 wt%, and mixing the almond husk and corn gluten in the same wt% ratio; And an ensiling step of storing the mixture in a silo container and sealing it.

According to the present invention, as a result of treating at least one selected from metabisulfite and organic acids in fruit and vegetable waste (FVD), the microbiological stability and freshness of FVD in an aerobic environment were maintained for a longer time, and the nutrients and antioxidant functions It was confirmed that it is very effective in preservation. In addition, the silage feed manufacturing method comprising a pretreatment step of treating at least one selected from metabisulfite and organic acids in an aerobic environment according to the present invention has the effect of improving the preservation properties during the anaerobic preservation process, especially almonds Co-ensiling of FVD with husk and corn gluten showed the best preservation properties.

1 shows the chemical composition and proportion (% DM, unless otherwise stated) of the fruit and vegetable waste used in Experiments 1 and 2 of Example 1 (mean±SD, n=5).
Figure 2 shows the moldy appearance, putrid odor, pH, and water-soluble carbohydrate (WSC) content of FVD according to the SMB throughput and time (Experiment 1 of [Example 1]).
3 shows the total number of bacteria (A), fungi (B), and yeast (C) as a function of SMB throughput and time (Experiment 1 of [Example 1]). Error bars at each point represent SE. Numerical values are expressed as the average of the five observations. The number of microorganisms is the logarithm number of colony-forming units/g wet biomass. The detection limit was 2.70.
4 shows the chemical composition of FVD as a function of SMB throughput and time (Experiment 1 of [Example 1]).
5 shows the pH, WSC, and ammonia content (NH ₃ -N) of FVD at different locations affected by SMB treatment.
6 is a result of analysis of microorganisms at different locations affected by the SMB treatment (Experiment 2 of [Example 1]). A) after 6 days; B) After 9 days. The total number of bacteria, yeast and fungi on day 0 for the control group averaged 5.97, 3.86, and 4.03 log cfu/g biomass, respectively. The total number of bacteria, yeast, and fungi on day 0 for the SMB treatment group was 4.26, <2.70, and 2.83 log cfu/g biomass, respectively. Values are expressed as the average of five observations. The number of microorganisms is the logarithm number of colony-forming units/g wet biomass. The detection limit was 2.70. Error bars at each point represent SE.
Fig. 7 shows the nutrient profile of FVD at different locations affected by SMB treatment (Experiment 2 of [Example 1]; mean±SD).
Figure 8 shows the microbiological, physicochemical properties and antioxidant capacity of liquid FVD affected by SMB treatment after 6 and 9 days.
Figure 9 shows the chemical composition and ratio of the total mixed waste used in silage production (mean ± SD, n = 4).
Figure 10 shows the composition of the composition of the silage (Experiment 2 of [Example 2])
Figure 11 shows the nutrient composition and microbiological properties of FVD in the presence or absence of SMB after storage for 0 and 7 days under aerobic conditions (mean ± SD, n=4).
12 shows the microbiological and physicochemical properties of FVD with or without SMB treatment according to anaerobic storage period (mean ± SD, n=4). The number of microorganisms is the logarithm number of colony-forming units/g fresh matter. The detection limit was 2.8 log cfu per g fresh matter. The ensiling for 0 days corresponds to the end of the retention period (7 days; see Fig. 11).
13 shows the change in total DM loss during the 90-day ensiling process. a) Silage without moisture control in the presence or absence of metabisulfite (Experiment 1 of [Example 2]). b) Moisture controlled silage with different SMB throughput (Experiment 2 of [Example 2]). Total DM loss was calculated from the deviation between weight and DM content. SMB0 = control silage without metabisulfite added. The proportion of FVD in the control silage was 66.6%. Error bars at each point represent SD. SMB = sodium metabisulfite.
14 shows the passage of free sulfite content over time. a) Silage without moisture control in the presence or absence of metabisulfite (Experiment 1 of [Example 2]). b) Moisture controlled silage with different SMB throughput (Experiment 2 of [Example 2]). Day 0 corresponds to the end of aerobic storage (7 days). The detection limit was 2 mg/kg DM. Error bars at each point represent SD. SMB = sodium metabisulfite.
15 shows the nutrient composition of silages having different SMB throughput during each storage period.
16 shows microbiological and physicochemical properties of silages having different SMB throughput during each storage period.
17 is a result of chemical and microbiological analysis for evaluating the aerobic stability of silage after 90 days of ensiling and 5 days of aerobic exposure.

The present inventors conducted two experiments using sodium metabisulfite (SMB) to investigate the effect of metabisulfite on the preservation of fruit and vegetable waste (FVD) in an aerobic environment (Example One). First of all, in the first experiment, metabisulfite was treated in amounts of 0, 2, 4, 6, and 8 g/kg FVD for 0, 3, 6, 9, and 12 days. As a result, 6 and 8 g/kg FVD It was confirmed that metabisulfite treated with sheep was most effective in preserving nutrients and inhibiting spoilage for 6 and 9 days, respectively. The second experiment was a pilot scale experiment where 0 and 8 g/kg FVD of SMB was treated for 0, 6, and 9 days in a 600-L bucket in an outdoor environment. The SMB treatment was very effective in maintaining the integrity and freshness of FVD, inhibiting microbial growth, and preserving nutrients. According to the embodiment of the present invention, SMB can effectively preserve FVD in an aerobic environment, and thus, FVD can be used as feed through long-term recycling.

Furthermore, the present inventors conducted two experiments to evaluate the storage stability of FVD through ensiling based on the experimental results (Example 2). In this experiment, FVD was not treated with SMB, or 6 g SMB/kg FVD was treated and pretreated in an aerobic environment for 7 days. In the first experiment, FVDs treated with SMB-treated with high moisture content biomass were ensiled alone. The SMB-treated silage had a low microbial population and negligible nutritional losses. In the second experiment, SMB-treated FVD was co-ensiling with almond husk and corn gluten at various rates, in which case the calculated SMB load was 4, 3.2, 2.4 and 1.6. It was g/kg biomass. As a result of microbiological analysis, nutritional characteristics, and sensory evaluation, it was evaluated that the SMB throughput of 3.2 g/kg biomass showed the most desirable preservation characteristics.

Furthermore, the present inventors treated SMB and organic acid based on the above experimental results and pre-treated in an aerobic environment for 7 days. As a result, the loss of dry matter and water-soluble carbohydrates was insignificant in the treatment, and the combination of SMB and organic acid was effective in promoting the growth of useful microorganisms, inhibiting the growth of harmful bacteria, and preserving nutrients.

Accordingly, the present invention can provide the use of metabisulfite as a composition for preserving feed raw materials that can improve the preservation properties of fruit and vegetable waste that can be used as feed raw materials, and based on this, fruits and vegetables in an aerobic environment It is possible to provide a method for producing a recycled feed for fruit and vegetable wastes, in particular silage feed, having improved preservation properties, including the step of pretreating metabisulfite to vegetable waste.

Hereinafter, the present invention will be described in more detail by examples. The following examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

[Example 1] Effect of SMB (sodium metabisulfite) treatment on FVD (fruit and vegetable discards) preservation

Experiment method

1-1. Sample preparation and experiment design

The first experiment (Experiment 1) consists of 25 combinations. Briefly, FVD (approximately 600 kg) was obtained for 3 consecutive days at a commercial packing house (E-mart Fresh Center, Icheon, Korea). The wastes were crushed into pieces of approximately 30-40 mm, mixed thoroughly and then randomly divided into 5 sub-samples. Metabisulfite powder was not added, or 2, 4, 6 or 8 g/kg wet FVD was added. Using a feed mixer (DDK-801M, Daedong Tech, Korea), the ingredients in each bucket were thoroughly mixed with SMB for 10 minutes, and each treatment group was randomly mixed into 5 replicate buckets. Each bucket was divided into 4 kg of FVD. Accordingly, a total of 125 buckets (10L) were prepared. The bucket was placed for 0, 3, 6, 9 and 12 days in an aerobic environment such as ambient temperature. At these time points, the total components contained in each bucket were thoroughly mixed and representative sub-samples were obtained at random locations for chemical and microbiological analysis.

In the second experiment (Experiment 2), FVD (about 2500 kg) was obtained for 3 consecutive days in the same place as above. Metabisulfite powder was not added or was added in an amount of 8 g/kg wet FVD. After the FVD was pulverized and thoroughly mixed in the same way as above, each treatment group was randomly distributed into 4 replicate buckets to contain 300 kg of FVD in each bucket. To minimize sampling errors, each FVD 2-kg allotments was placed in a mesh sack (280 mm wide x 560 mm long, 1 mm2 void). The sacks were placed in three different locations in each bucket: surface (0-5 cm deep), middle (25-40 cm deep) and bottom (55-70 cm deep). The bucket (600-L volume; 102 cm high; 97 cm wide) was placed under outdoor aerobic exposure conditions for 0, 6 and 9 days. The maximum, minimum and average temperatures during the experiment were 28.4, 22.5, and 25.1°C, respectively. At the designated time for each treatment group, all contents in each bag were collected and thoroughly mixed, and representative samples were randomly selected for analysis.

1-2. Visual, microbiological, and physicochemical characterization

Moldy appearance evaluation was scored as 0 if the mold was not visually evident, and 1 if it appeared clearly (Kwak, WS, et al., 2008. Broiler litter supplementation improves storage and storage and). feed-nutritional value of sawdust-based spent mushroom substrate.Bioresource Technology 99, 2947-2955). The absence and presence of a decaying odor were scored as 0 and 1, respectively (Kwak et al., 2008). Three panels performed these visual and sensory evaluations.

To investigate the metabolites produced during the storage period, 20 g of each sample was mixed with 80 mL of deionized sterile water in a sterilized plastic bottle to obtain a juice extract. The suspension was shaken for 10 minutes and then filtered through two layers of cheesecloth. The pH of the extract was measured with a portable electrode (HI9321, Hanna Instrument, Portugal), and microorganisms were counted. Total bacteria and lactic acid bacteria (LAB) were spread-plated on plate count agar and MRS agar (de Man-Rogosa-Sharpe agar) (Difco Laboratories Inc., Detroit, MI, USA), respectively. It was counted using. The plate was incubated at 36 ± 1 °C for 48 hours (HK-IB157, Korea Total Instrument, Korea). Yeast and mold were visually identified and counted in yeast extract glucose chloramphenicol agar (Difco Laboratories Inc.) by spread-plating method. The plates were incubated at 25±1° C. for 3 days for yeast and 5 days for mold. The detection limit was 2.7 log cfu/g. Using S-1100 UV spectrophotometer (Scinco, Republic of Korea), water-soluble carbohydrates (WSC) were determined by phenol-sulfuric acid procedure (Dubois, et al., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350-356), NH3-N was quantified by a phenol-hypochlorite procedure (Chaney and Marbach, 1962. Modified reagents for determination of urea and ammonia. Clinical Chemistry 8, 130-132). According to the standard analysis method of AOAC (The Association of Official Analytical Chemicals, 2012), DM (Analysis Method No. 930.15), ether extract (Analysis Method No. 2003.05), crude protein (N × 6.25; Assay No. 990.03), ash ( Assay No. 942.05), NDF (neutral detergent fiber) (containing heat-resistant α-amylase and sodium sulfite; Assay No. 2002.04), and ADF (acid-detergent fiber) (Analysis method No. 973.18) were measured. The non-fibrous carbohydrate (NFC) was calculated in the formula of 100-[crude protein + NDF + ether extract + ash].

One- 3. 2 ,2- Diphenyl -One- picrylhydrazyl Assay

A 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay was performed on the liquid phase formed in the process of leaking juices and sugars from the cut FVD tissue of Experiment 2. Experimental methods are known methods (Wootton-Beard et al., 2011. Stability of the total antioxidant capacity and total polyphenol content of 23 commercially available vegetable juices before and after in vitro digestion measured by FRAP, DPPH, ABTS and Folin-Ciocalteu methods. Food Research International 44, 217-224). Samples were centrifuged at 4000×g at room temperature for 5 minutes, and the supernatant was collected. A methanol solution of DPPH. (0.1 mM, 3.9 mL) was added to a 50-μL aliquot of the supernatant. The mixture was allowed to stand for 30 minutes at room temperature under dark conditions. The absorbance was measured at 517 nm with an S-1100 UV spectrophotometer (Scinco, Korea). The percent inhibition of DPPH radicals was calculated for the initial DPPH absorbance (without sample).

1-4. Data analysis

The data of Experiment 1 were analyzed using PROC MIXED with a completely randomized design. The treatment groups were arranged in a 5×5 factorial design to create a total of 25 combinations. The separation of least-square means was performed using Tukey's range test. The data in Experiment 2 were applied with ANOVA and the deviation between treatment group means was confirmed using Student's t -test. The replicate bucket was considered an experimental unit. Microbial counts were converted to logarithm numbers of colony-forming units prior to statistical analysis. P <0.05 was considered a significant value.

Experiment result

1-1. Initial characterization

Components and chemical compositions of FVD used in Experiments 1 and 2 are shown in FIG. 1. The proportion of each component of the total waste was calculated as the average monthly production of waste in the month of the end of the experiment. The above major components accounted for more than 95% of total waste per month. In each of Experiments 1 and 2, oranges and plums accounted for the largest proportion of the total waste mixture. Among the analyzed components, sweet pumpkin and potato showed the highest DM content, and tomato and onion showed the lowest DM content. The content of crude protein was highest in green onion stems and lemons, while grapes and apples had the lowest ash content. Ether extract was negligible in potatoes and was the highest in sweet pumpkin and chive stems. The NDF content was highest in sweet pumpkin, followed by paprika and chive stems, and the lowest in grapes and potatoes.

1-2. Experiment 1: Visual, microbiological and chemical characterization

As a result of examining the appearance of FVD treated with or without SMB during the experiment, it was confirmed that FVD treated with 6 and 8 g SMB/kg biomass maintained freshness without damage until 9 days later. In general, FVD without any treatment gradually develops browning. However, as the SMB load increased, the color change decreased, because SMB suppresses the browning reaction. Sulfites readily react with carbonyl intermediates to block the formation of brown pigments during non-enzymatic browning. In addition, sulfite inhibits the activity of many enzyme-catalyzed reactions, especially polyphenol oxidase, a catalyst that oxidizes phenolic compounds to quinones. Quinones play a key role in the production of brown pigments in damaged tissue during the enzymatic browning process. In addition, sulfite treatment played a role in maintaining the integrity of the tissue. FVD without SMB treatment is rapidly softened because the cell wall is degraded by enzymes secreted from the damaged tissue.

Fig. 2 shows the moldy appearance and decaying smell of FVD according to SMB throughput and time. After 3 days, mold appeared on the surface of the waste with a decaying odor in the control sample, and in the sample treated with the least SMB (2 g/kg biomass), it was found to be less than the control sample. This shows that waste biomass decays very quickly. The samples treated with the most SMB did not smell of decay until the 9th day, which means that microorganisms causing decay were suppressed. In addition, since sulfite has the effect of inhibiting the oxidation of carotenoids and essential oils, which are related to the occurrence of undesirable odors, it partially explains the phenomenon that the occurrence of odors in SMB-treated FVD is suppressed. can do.

In the sample treated with the most SMB, mold development and decay were effectively delayed for 9 days, but after 12 days, a slight decaying odor was observed, possibly due to the gradual loss or inactivation of SMB during the preservation period. Metabisulfite is a strong reducing agent that is oxidized when exposed to oxygen, reducing its preservative effect. Overall, it has been shown that treatment of 6 and 8 g/kg biomass of SMB effectively prevents FVD spoilage and mold formation until days 6 and 9, respectively.

Figures 3A-C show the total number of bacteria, mold and yeast over time and SMB load during aerobic storage of FVD. Compared with the number of microorganisms detected on day 0 (before the treatment of SMB), the number of microorganisms in the sample treated with the largest number of SMBs (8 g/kg FVD) was significantly reduced, and this resulted in the inhibitory effect of SMB on the microorganism. Because it appeared immediately. The microbial populations detected on day 0 represent microbes that occur and reside during the process of harvesting, distribution, transport, storage and packaging of fruits and vegetables.

In the control sample, total bacteria increased rapidly, and viable bacteria reached 7.79 log cfu/g after 3 days. However, SMB treatment of 6 and 8 g/kg biomass was very effective in inhibiting the growth and proliferation of total bacterial individuals even after 9 days. Similarly, in the control sample, yeast and mold individuals significantly increased as the duration of the experiment increased (after 6 days). However, in the sample treated with the most SMB, the growth of yeast and mold was very effectively inhibited. Although the mechanism for inhibiting microbial activity has not yet been identified, it is believed that this is because sulfiting agents exhibit toxicity to enzymes and inhibit enzymes essential for microbial growth. It is also believed that the strong reducing power of SMB was toxic to aerobic organisms and some enzyme systems.

The pH and WSC content of FVD according to SMB throughput and time are as described in FIG. 2. During the experiment, the pH of the SMB-treated (especially, 6 and 8 g SMB/kg biomass-treated FVD) remained relatively constant, but decreased by about 0.6 units after 9 days in the control sample. I did. This shows that FVD is effectively preserved by SMB processing. In the FVD sample not treated with SMB, the WSC content gradually decreased (from 63.5 g/100 g DM on day 0 to 12.7 g/100 g DM on day 12). However, in the samples treated with SMB of 4-8 g SMB/kg FVD, the WSC content decreased to a negligible level during the experiment.

The decrease in pH and WSC according to the progression of decay in the control sample may be due to the use of WSC according to the rapid increase of microorganisms (FIG. 3 ). In general, pH is an indicator of acid production and decay during the aerobic decay process. If the acid production rate exceeds the decay rate, a decrease in pH is usually observed during the aerobic decay process.

1-3. Experiment 2: chemical composition analysis

The nutrient composition profile of FVD according to SMB throughput and time is shown in FIG. 4. As the SMB throughput increased, the ash content increased from 4.50% to 7.61%, which is related to the high ash content of SMB (71.7%, Fig. 1). As the experiment progressed, the nutrient profile of FVD without SMB or 2 g of SMB was gradually changed compared to other treatment groups. Compared with the 0 day, the NFC content of the control sample decreased by 30.2% after 12 days, which means the accumulation of NDF, protein, and ether extract and ash. This is evidenced by the increase in NDF and ADF content of aerobicly unstable corn silage in other studies, which can be explained by the depletion (use) of WSC and the resistance of the fiber fraction to aerobic spoilage. However, the NFC content of the SMB-treated sample (4-8 g) was not significantly affected during the storage period, suggesting the effectiveness of SMB in maintaining the easily oxidized components in FVD. The high content of NFC in FVD means that it can be considered as a partial substitute for food with high energy density.

The longer the storage period, the greater the DM loss in the control sample (22% decrease after 12 days), which may be due to the conversion of carbohydrates to water and carbon dioxide during the aerobic digestion process. Studies on aerobic decay in silage show that high yeast populations contribute to the rapid conversion of soluble carbohydrates into ethanol, carbon dioxide and water, resulting in significant DM losses. The large number of yeast in the control sample can contribute to rapid depletion of WSC and DM loss (Fig. 3C).

The crude protein content was not different in FVD treated with 4-8 g SMB/kg biomass. However, in the control sample and FVD treated with 2 g SMB/kg biomass, gastric components increased during the experiment. Conversely, the DM content of FVD treated with 4-8 g SMB increased during the experiment period, which may be related to its surface dehydration. However, despite the continued increase in microbial growth and accelerated decay, loss of nutrient composition in FVD treated with 4 g SMB was negligible even after 12 days. Overall, SMB treatment of 6 and 8 g/kg biomass was very effective in preserving nutrients of FVD even after 12 days of aerobic storage. Based on microbiological and sensory evaluation, SMB treatment of 6 and 8 g/kg biomass during aerobic exposure provides effective preservation for at least 6 and 9 days, respectively.

1-4. Experiment 3: microbiological characterization

In this pilot scale experiment, the inventors obtained sub-samples at different locations on the top, center, and bottom portions of each bucket. The physicochemical and microbiological properties of the waste samples obtained at the different locations were found to be very diverse. Therefore, the data of this experiment were not combined with each other between the locations. Also, several days after the FVD was transferred to the bucket, the substances began to flow out as liquid. Therefore, the present inventors used separated samples for analysis of liquid microorganisms.

Figure 6 shows microbial individuals of FVD at various locations affected by SMB treatment. Metabisulfite led to significant inhibitory effects in the microbial profile of FVD. Similar to Experiment 1, SMB treatment significantly inhibited yeast and mold individuals. Compared to the control, FVD treated with SMB (8 g) showed significantly less total bacteria, yeast and fungi even after 9 days. Mold growth was not evident in SMB-treated samples obtained from the center and bottom. Both the control and SMB-treated samples showed a greater number of total bacteria, yeast and fungi in the upper layer than in the central and bottom locations. Compared to the 6th day of storage, a logarithmic reduction of 0.36 and 0.61 log cfu/g was found in the total bacterial and yeast populations in the upper layer of the control sample after 9 days.

Changes in pH, WSC and ammonia concentration of FVD at the various locations affected by SMB treatment are shown in FIG. 5. Similar to experiment 1, the pH of FVD on day 0 was acidic, and slightly increased (0.18 units) after SMB treatment. Yeasts and fungi were believed to be the main causes of microbiological decay of FVD due to their ability to grow and dominate in low pH fruits. In general, metabisulfite ions in their undissociated form are toxic because they can penetrate into microbial cells. When the maximum concentration of the non-dissociated form reaches between pH 3-4, the increased hydrogen ion (H+) concentration will inhibit the dissociation of the sulfite molecule. In both experiments, the acidic pH of the initial FVD indicates that most of the SMBs did not dissociate and existed in a more potent bactericidal form.

Although there were slight differences in the pH values at different locations of the SMB-treated biomass, the pH value increased significantly in the upper layer sample of the control sample as the experiment period increased (from 3.67 on day 0 to 6.61 after 9 days). . This increase in pH is usually seen in silages that are decayed aerobicly because organic acids are oxidized to carbon dioxide and water, and at the same time the production of acidic end products due to the metabolism of aerobic microorganisms decreases.

Compared to the control, the WSC content was reduced to a much lower degree in the SMB-treated FVD, which means that the SMB treatment effectively inhibited the microorganisms associated with the rapid utilization of carbohydrates. However, as the experiment progressed, the WSC content decreased significantly in the control sample. For example, the WSC content on the upper surface decreased from the initial content of 60.1 g/100 g DM to 2.47 g/100 g DM on the 9th day. The reduction can be explained by the active growth and metabolic activity of aerobic microorganisms that can easily use carbohydrates on the upper surface (FIG. 6). In addition, the downward shift of the soluble component is in part associated with a decrease in the upper WSC.

The ammonia content increased as the experiment proceeded in both the control and SMB-treated samples. However, the increased amount was smaller in the SMB-treated sample, indicating the inhibitory effect of SMB on the microorganisms involved in proteolysis and ammonia production. After 6 days of aerobic storage, the ammonia content on the upper surface of the control sample increased from the initial value of 4.09 to 24.3 μg/g DM and decreased to 12.3 μg/g DM after 9 consecutive days. These losses can be explained by the conversion of ammonia nitrogen to nitrate nitrogen and then to nitrite nitrogen and nitrous oxide under an aerobic environment.

1-5. Experiment 4: Analysis of nutrient composition composition

Fig. 7 shows changes in the composition of FVD nutrients at different locations affected by SMB treatment. The same pattern as the nutrient composition pattern of waste observed in Experiment 1 was found in a pilot scale experiment. Similar to Experiment 1, SMB treatment was very effective in preserving the nutrient composition of the waste after 9 days. In general, various changes in chemical components were observed at different locations of the control sample, and the chemical components were remarkably changed on the upper surface. For example, after 9 days, the NFC content on the upper layer surface decreased from the initial value of 68.6% to 8.57%, and decreased to 32.5% in the bottom area. This decrease appears to be due to the rapid depletion of easily oxidized components, and the relatively increased proportions of more complex carbohydrates (NDF and ADF), crude protein, ether extract and ash.

The accumulation of fibrous fraction can be explained by its chemical complexity, which makes it resistant to aerobic decay. At the same time, readily available carbohydrates can be easily consumed by microorganisms. The fat component of the upper layer of the control sample was also significantly increased (from 1.65% on day 0 to 6.84% after 6 days of aerobic preservation), indicating the resistance of the fat component to aerobic decay.

1-6. Experiment 5: liquid Fraction Analysis of microbial and antioxidant properties

The microbiological profile of liquid FVD affected by SMB treatment after 6 and 9 days is shown in FIG. 8. The number of viable LABs, molds and yeasts was high in the control sample, but the microorganisms were not found at the detection level of 500 cfu/g biomass in the SMB-treated sample.

After 6 and 9 days, the WSC content in the SMB-treated biomass was significantly higher compared to the control, and this phenomenon can be explained by a significant reduction in microbial populations that readily consume WSC in the SMB-treated sample. Similar to the data on the ammonia content of FVD at other locations, a much lower concentration of ammonia was found in the SMB-treated sample compared to the control after 9 days of storage (31.1 vs. 63.3 μg/mL).

The SMB-treated liquid obtained after 6 and 9 days scavenged 26% and 31% more DPPH radicals, respectively compared to the control, indicating that SMB treatment preserved the antioxidant capacity of FVD. The reducing ability of the liquid fraction to DPPH radicals decreased slightly in both the control and SMB-treated samples as the experimental period increased from 6 to 9 days.

In conclusion, based on the above visual, microbiological and chemical evaluations, treatment with metabisulfite of 6 and 8 g/kg biomass upon aerobic exposure improved the stability and freshness of discarded fruits and vegetables for 6 days and 9, respectively. You can see that you can keep it for days. Pilot-scale experiments demonstrated that metabisulfite treatment for 9 days in an aerobic environment was effective in delaying spoilage, improving microbiological stability, and preserving nutrient and antioxidant functions.

[Example 2] Long-term preservation of FVD through SMB treatment

Experiment method

2-1. Sample preparation and preservation studies

Detailed procedures regarding the sample preparation and preservation process followed the previous research method of the present inventors. Briefly, FVD (about 600 kg) was obtained for 3 consecutive days at a commercial packing house (E-mart Fresh Center, Icheon, Korea). Information on the amount of individual ingredients disposed of each month was investigated through input and output data provided by the shipyard management department. In the month in which the experiment was conducted, major wastes, accounting for more than 90% of the total waste, were investigated and obtained at the shipyard facility. The chemical composition of the individual components is shown in FIG. 9.

The wastes were cut into approximately 30-40 mm pieces, thoroughly mixed and then randomly divided into 7 samples. Metabisulfite powder was added in an amount of 0 or 6 g/kg FVD. Using a feed mixer (DDK-801 M, Daedong Tech, Korea), the samples were thoroughly mixed for 10 minutes under conditions with or without SMB addition, and then transferred to a 200L bucket. To simulate the actual situation of FVD accumulation at the source, the bucket was placed in an aerobic environment such as ambient temperature for 7 days. Representative sub-samples were obtained at any location for chemical and microbiological analysis.

2-2. Silage manufacturing

After 7 days of exposure to an aerobic environment, SMB-treated FVDs were ensiled in silos with high humidity biomass alone or with almond husk and corn gluten supplied at different rates. The ratio of FVD in the silage according to this example was 66.6%, 53.2%, 39.8% and 26.6%, which is calculated to provide 4, 3.2, 2.4 and 1.6 g of SMB per 1 kg of biomass, respectively (Fig. 10). .

Silage with 66.6% FVD content without SMB was used as a control. FVD was ground using an electronic grinder to minimize the difference in particle size. The ground FVD without a moisture absorbent was stored (ensiled) in a 3L glass bottle silo weighed in advance. Four glass bottles were used for each treatment.

The rest of the ground FVD was mixed with different ratios of almond husk and corn gluten feed using a feed mixer (DDK-801 M, Daedong Tech, Korea). The characteristics of FVD biomass are high content of simple carbohydrates, high moisture content, and low protein content. Therefore, considering the nutritional balance of the ensiling mixture as well as water-holding power and economic utility, almond husk as a byproduct of a moisture absorbent rich in fiber and corn gluten as a byproduct of a moisture absorbent rich in energy and protein are ensiling together with FVD. It was selected as an ensiling companion. The mixture (about 4 kg) was packaged in a polyethylene bag (45 cm wide x 75 cm high), tightly sealed, and ensiling at room temperature (20-25° C.). The silos were weighed on the pre-selected opening days (7, 14, 30, 60 or 90 days), and representative subsamples were randomly obtained for nutritional and microbiological evaluation. Silage was evaluated visually and sensory by three experienced assessors.

2-3. Microbiological evaluation and analysis method

Fermentation metabolites and microbial populations were investigated in the silage extract obtained by mixing a silage sample (20 g) with 120 mL of autoclaved distilled water. The suspension was shaken for 5 minutes and then filtered through two layers of medical gauze. The pH of the silage extract was immediately measured with a portable electrode (HI9321, Hanna Instrument, Portugal). For the analysis of water-soluble carbohydrates (WSC; glucose equivalent; Dubois et al., 1956) and NH3-N (Chaney and Marbach, 1962), another portion of the filtrate was centrifuged at 4° C. at 10,000 g for 10 minutes. I did.

Ammonia-N was presented on an N equivalent basis. After appropriate serial dilution of the silage extract, the microbiological composition of the silage samples was analyzed using a spread-plating method. The total number of viable bacteria was counted on a plate count agar (Difco Laboratories Inc., Detroit, MI, USA). Viable colonies of lactic acid bacteria (LAB) were counted in MRS agar (Difco Laboratories Inc., Detroit, MI, USA). The plate was incubated at 36° C.±1° C. for 48 hours (HK-IB157, Korea Total Instrument, Korea). Yeast and mold were counted by yeast extract glucose chloramphenicol agar (Difco Laboratories Inc.). For counting, the plates were incubated at 25° C.±1° C., 3 days for yeast and 5 days for mold. Differentiation of yeast and fungi was achieved by visual evaluation of colony appearance.

For nutrient analysis, a fresh sample (about 200 g) was dried in a forced-air oven (72 hours at 65° C.) and then pulverized to a particle size of 1 mm. According to the standard analysis method of AOAC (The Association of Official Analytical Chemicals, 2012), DM (Analysis Method No. 930.15), Ether Extract (Analysis Method No. 2003.05), Crude Protein (Analysis Method No. 990.03), Crude Ash (Analysis Method) No. 942.05), NDF (neutral detergent fiber) (containing heat-resistant α-amylase and sodium sulfite; Analysis Method No. 2002.04), and ADF (acid-detergent fiber) (Analysis Method No. 973.18) were measured. The non-fibrous carbohydrate (NFC) was calculated in the formula of 100-[crude protein + NDF + ether extract + ash].

The time course of total DM loss was calculated from the difference between the initial weight (day 0) and days ensiling 7, 14, 30, 60 and 90, based on the DM content of the silage mass. The free sulfite content of the SMB-treated sample was quantified according to an enzymatic colorimetric method using a commercial kit (K-SULPH kit, Megazyme International Ireland Ltd., Bray, Co., Wicklow, Ireland). The aerobic stability of the final silage (90 days) was determined using a bottle system that measures the amount of carbon dioxide produced during aerobic deterioration (Ashbell et al., 1991). The silage was exposed to aerobiosis for 5 days at room temperature (25°C). The amount of carbon dioxide produced after aerobic deterioration, pH value, total number of bacteria, yeast and mold were used as indicators of aerobic spoilage.

2-4. Statistical analysis

Microbial data was converted to log ₁₀ prior to statistical analysis. Conservation studies of Experimental Method 1 and statistical analysis of silage were performed using a general linear model of SAS (SAS, 2004). When the F value was significant, the deviation between the least-square means was confirmed using a t -test at the 0.05 probability level. The rest of the data was analyzed using PROC MIXED with a completely randomized design in SAS (SAS, 2004). The model below was used for analysis.

Y _ij =μ+T _i +ε _ij

Where Yij is the observation j in the treatment i, μ is the average, Ti is the storage period or the fixed effect of the treatment (aerobic stability test), and ij is the residual error. Polybag Silo was considered an experimental unit. When a significant treatment effect was found (P <0.05), separation of least-square means was performed using Tukey's test. The linear response to the storage period from 0 to 90 days was estimated using polynomial contrasts (SAS, 2004).

Experiment result

2-1. Aerobic preservation

Fig. 11 shows the nutrient composition and microbiological properties of FVD in the presence or absence of SMB after storage for 7 days in an aerobic condition. Overall, SMB was very effective in minimizing nutrient loss and reducing the number of bacteria, yeast and fungi. LAB was found to be very susceptible to SMB and was not found at a detection level of 2.8 log cfu/g in SMB-treated samples after storage for 7 days. LAB is known to produce various amounts of hydrogen peroxide, which reacts strongly with sulfite to produce sulfur trioxide anion radicals. These radicals are known to strongly inhibit the cell growth of LAB. In addition, the unpublished preservation study results of the present inventors also showed a strong inhibitory effect of SMB on LAB, yeast and mold.

After storage for 7 days in aerobic conditions, the WSC content in the control sample was significantly reduced, but crude protein (CP), ash, and NDF were accumulated. This increase can be explained by the decrease in organic matter during the preservation process of FVD under aerobic conditions, which converts carbohydrates into carbon dioxide and water. As in Example 1 above, the present inventors performed a series of storage experiments with SMBs of various loadings at the laboratory and pilot scale, and as a result, 6 and 8 g/kg wet masses under aerobic exposure environment. It has been found that SMB keeps FVD relatively stable and can prevent loss of nutrients for up to 6 and 9 days, respectively. Therefore, the purpose of the experiments according to this embodiment was to extend the shelf life of FVD for a longer period through ensiling.

2-2. Experiment 1: anaerobic storage without moisture control

Microbiological and physicochemical properties of FVD with or without SMB treatment according to the anaerobic storage period are shown in FIG. 12. The development of viable LAB reached a peak in 7.67 log ₁₀ cfu/g biomass after 14 days of ensiling period, and decreased with time in control silage, then 5.96 log cfu after 90 days of fermentation. /g reached biomass. This can be seen as a general result because previous studies have reported that the number of LABs decreases as the ensiling duration progresses. However, no LAB was found in the SMB silage (limit of detection = 2.8 log cfu/g). For efficient anaerobic conservation, lactic acid fermentation must take place quickly. LAB is known to be a major consumer of WSC during ensiling by converting WSC to lactic acid and promoting a rapid decrease in silage pH.

In this example, it was confirmed that the rapid development of LAB immediately reduced the pH of the control silage (FIG. 11). However, a decaying odor was detected as the number of unwanted microorganisms (i.e. yeast and mold) increased in the silage mass. According to previous studies, depending on the microflora of the pre-ensiling biomass, even if the number of LABs increases rapidly, it inhibits undesirable microbial growth or prevents DM loss during ensiling. It may not be possible. Essentially, lactic acid is a weak fungicidal compound, so if the number of undesirable microorganisms is high, it will not be strong enough to prevent their growth. In the embodiment of the present invention, even though the LAB rapidly proliferated, the growth of undesirable microorganisms was not inhibited during ensiling, so the above phenomenon may also occur in this example. Unlike the control silage, the LAB decreased below detectable levels in the SMB-treated silage during the 90-day storage period, but the loss of nutrition was negligible with respect to the low number of undesirable microorganisms. These results suggest that when FVD is ensiling without an absorbent, SMB is more effective as a silage fermentation inhibitor than hydrogen ions.

During the storage period of 90 days in the SMB-treated silage, the number of molds was below the limit of detection, but the yeast was at a low level (less than 4 log cfu/g) during the initial stages of ensiling. It was not detected in SMB silage after days and 90 days of storage. Some of these observations may be related to the provision of stringent anaerobiosis by SMBs in the early stages of storage.

When SMB is used as a silage additive, it breaks down into sulfur dioxide, a strong antifungal compound that consumes significant amounts of oxygen during ensiling. This reduces aerobic stress during the initial stages of the ensiling process, thus providing effective inhibition against aerobic microorganisms. Although the antimicrobial mechanisms of sulfiting agents are not clearly understood, due to their strong nucleophilicity, sulfites appear to react easily with biomolecules through substitution reactions at the electrophilic site. It initiates a series of reactions that can damage key enzymes involved in the metabolism of microbial cells.

During 90 days of ensiling, the NH ₃ -N/N content in both the control and SMB silage was lower than the total N threshold of 70 g/kg, which is estimated to be an indicator of successful silage fermentation. The low ammonia content of the control silage may be associated with a rapid decrease in pH, which is known to minimize protein degradation.

In addition, the total DM loss during the storage process is shown in Fig. 13. A major DM loss was seen in the control silage, and 21.1% was lost during the 7-day ensiling period. A similar trend was observed in the WSC content, which decreased from 29.7% on day 0 to 16.6% after 7 days of ensiling. However, after 30 days of ensiling, the silage fermentation appeared to have stabilized. Unlike the control silage, a small loss of DM and WSC was observed in the SMB-treated silage (DM decreased from 15.3% on day 0 to 14.3% after 90 days).

The strong microbial population present in the control silage mass prior to ensiling contributed to the complete oxidation of organic matter such as WSC and fermentation products to carbon dioxide and water, which is associated with excessive loss of DM and nutrients. This represents a potential loss of feed nutrients available to the animal and is an undesirable waste of energy that should be avoided. In general, conservation through ensiling is typically in the typical range of 15%-20%, and is primarily related to the energy cost of DM losses (known as'silage reduction') resulting from carbon dioxide production, It has environmental and economic importance. This DM loss increases even more when biomass with high simple sugar content and low DM content is ensiling. Consequently, the factors contributing to the excessive loss of nutrients in the control silage are probably the following: 1) aerobic exposure of FVD prior to ensiling, which increases the number of decaying microorganisms; 2) high moisture content of FVD; And 3) high WSC content of FVD.

Regarding the storage period, the course of the sulfite concentration released from the SMB silage with time is shown in FIG. 14. Compared to day 0 of storage, about 46% of the free sulfite was lost after 90 days of storage. This may help to partially explain the slight increase in microbial populations as ensiling proceeds in SMB-treated silage. In principle, when sulfite is used as a preservative, it will exist in free and combined form, and an equilibrium will be reached between the two forms. Of these, only the free form exhibits activity and functions as a preservative.

Overall, although the rapid development of LAB contributed to the rapid decrease in pH, the control silage exhibited poor preservation properties with an undesirable microbial profile and excessive DM loss, which resulted in silage from FVD not treated with effective preservatives. It is a factor that hinders the possibility of commercialization of production. However, in this example, it has been demonstrated that FVD can be treated with SMB and satisfactorily stored under anaerobic conditions. Metabisulfite alone can provide protection against undesirable microflora, which minimizes the loss of DM and nutrients, thereby providing a cleaner way to use the silage more safely over the long term. On-site anaerobic storage of soil FVD after treatment with SMB in oxygen-restricted storage tanks can facilitate the practical use of this conservation technique for continuous use as a livestock feed source. One of the main difficulties in standardizing FVD, an ingredient that is consistently and continuously used in livestock feed, is the diversity of ingredients and nutrient composition. If possible, storing relatively large amounts of FVD anaerobicly in the field, less than one month old, can minimize this diversity and better manage this waste as an ingredient for inclusion in animal feed.

2-3. Experiment 2: moisture controlled anaerobic storage

Prior to describing the results of this example, the present inventors attempted to make silage by adding almond husk and oat husk to SMB-treated FVD in an initial experiment (66% FVD, 17% almond husk, 17% oat husk. ; 42% DM). According to the results of a 90-day experiment, the silage began to decay after ensiling began. This was accompanied by loss of DM, nutrients and a sharp increase in the number of yeast and fungi. During the ensiling period, a slight decaying odor was detected along with a prevalent alcohol odor. Similar to Experiment 1 above, LAB was below the detection limit at all sampling times. It is speculated that the resulting increased porosity due to the low packing density and the addition of oat husks has resulted in a rapid increase in aerobic microorganisms, especially yeast and fungi. It is possible that the hollow structure of the oat shells increased the amount of air in the silage mass and thus increased the activity of aerobic microorganisms in the early stages of ensiling. It also appeared that the SMB load of the silage output was not sufficient to adequately suppress yeast and mold development. This led to the rapid conversion of readily available carbohydrates to carbon dioxide and water, which is associated with inefficient anaerobic conservation. Based on this assumption, the present inventors provided good compression with minimal air volume in the silage mass by selecting corn gluten feed with almond husk and small particle size in subsequent experiments.

Changes in the chemical composition and conservation characteristics of silages with different SMB loads are shown in FIGS. 15 and 16 according to the storage period (0, 7, 14, 30, 60 or 90 days). With the extension of ensiling, the ash content increased slightly in the SMB silage, and more in the control silage. As expected, the NFC content decreased significantly in the control silage and to a lesser extent in the SMB silage, indicating an increase in fermentation, oxidation of well-oxidized components, and more recalcitrant components such as fibers in the silage. It is an indicator. In normal silage manufacturing, delays in sealing (aerobic exposure) are generally associated with significant loss of nutrients and DM during ensiling. The on-site accumulation of FVD is a potential vector of many undesirable microorganisms. Therefore, the undesirable microbial profile (termination of aerobic preservation) of FVD prior to ensiling could contribute to the occurrence of potent metabolic processes during anaerobic storage in the control silage, which appears to have increased DM and nutrient loss.

In contrast, the nutrient loss of silages with higher SMB throughput showed subtle differences during 90 days of anaerobic storage. For example, in the silage of 3.2 g SMB/kg biomass, the WSC concentration was 21.8% before ensiling and decreased to 19.0% DM after 90 days of ensiling. To the best of the present inventors' knowledge, no studies have yet been made to investigate the anaerobic storage dynamics of FVD treated with SMB beforehand and aerobically challenged.

In the SMB silage, the ammonia level increased as the storage time increased, and the highest value was observed between 60 and 90 days of ensiling. This indicates that long ensylation times promote protein degradation. During the ensylation period, the protein can be degraded to ammonia, indicating clostridial fermentation of the protein. In general, an indicator of successful silage fermentation is an NH ₃ -N content below the 70 g/kg total nitrogen threshold, whereas an amount greater than 100 g/kg total nitrogen indicates poor fermentation of the silage. As can be seen from FIG. 16, it can be seen that there is no SMB silage having an NH3-N/N content higher than a threshold indicating high-quality silage.

Similar to Experiment 1 above, the LAB number was very low or below the detection limit at any SMB silage during the storage period. In general, total bacteria, yeast and fungi had the smallest populations in the silage with the highest FVD rate (higher SMB throughput). Although the LAB proliferated rapidly and dominated the silage fermentation (Figure 16), the pH of the control silage did not change during the 90-day ensiling process. It is believed that decaying or moldy silage prevents silage pH from decreasing during silage fermentation. In the embodiment of the present invention, the possibility that the above phenomenon may have occurred can be considered from the fact that the number of yeast and mold was large in the control silage accompanied by the smell of decay. In the case of metabisulfite silage, the driest silage (lower SMB throughput) showed an increase in silage pH as the ensiling period increased. In general, an increase in silage pH during the ensiling period indicates limited lactic acid fermentation, which is associated with undesirable microbial distribution and extensive nutritional losses in traditional silages. However, in these silos, the SMB load was probably sufficient to inhibit LAB growth, but as the ensiling period prolonged, the active form of SMB decreased to a level that allowed control of undesired microbes. Conversely, in the silage with the highest ratio of FVD (i.e. 4 and 3.2 g SMB/kg biomass), the silage pH was largely constant (about 4), and decreased slightly with prolongation of ensiling.

In general, a suitable preservation criterion for ensiled biomass is a target pH of less than 4, at which most undesirable bacteria are inhibited. The bisulfite ions themselves provide efficient preservation as opposed to the hydrogen ions (low pH), which are responsible for preservation in conventional silages. The above results support the conclusion that using SMB as a silage fermentation inhibitor is very effective in providing silage with good smell and high nutritional value.

As shown in FIG. 16, since the pH of FVD was acidic, the dissociation of bisulfite ions, which is more toxic to microorganisms when present in an undissociated form, was suppressed. Therefore, it is expected that more effective inhibition by SMB will be achieved in the natural acidity of FVD. Overall, metabisulfite silage provides a new type of silage that is not preserved through hydrogen ions, as occurs in conventional silage fermentation. Instead, in this type of silage, preservation is mediated by metabisulfite ions, contributing to the effective inhibition of microorganisms and reducing ammonia and acid formation during the ensiling process.

The total amount of DM loss of the moisture-controlled silage according to the storage period is as shown in FIG. 13. The loss of DM in the control silage was significant, and about 14.8% of the total silage DM was lost within 30 days of ensiling. However, after that, the rate of disappearance decreased, and after 90 days of ensiling, it only increased to 17.6%. These observations indicate that the metabolism of WSC and other components continues until the silage pH drops to the point where microbial activity slows and silage fermentation stabilizes, the fermentation period and DM loss during ensiling, controlled by the silage microbiota. It is consistent with the result of meta-analysis that found the relationship between the two.

Unlike the control silage, in the metabisulfite silage, negligible DM loss was found within 30 days after ensiling, but it started to increase slightly thereafter. Among metabisulfite silages, 3.2 g SMB/kg biomass had the lowest total DM loss, and only 3.98% of DM was lost in 90 days of ensiling. This suggests that the metabisulfite load and water content in this silage provide efficient preservation under anaerobic conditions for long periods of time without the need for lactic acid fermentation. Co-ensiling of SMB-treated FVD with almond husk and corn gluten feeds to approximately 50% is an effective way to control silage rot and possible transport difficulties associated with transporting moist materials. Can be seen, thereby increasing the usefulness of the silage and periodically transporting it to a distant location.

2-4. Visual and sensory examinations

Within 7 days of storage, the control silage began to decay, mold was seen on the surface, and a slight decayed odor was detected. Although no moldy spots were seen on the silage surface with the highest FVD ratio, mold formation was remarkable on the silage surface after 1 month of ensiling with a theoretical SMB load of 2.4 or 1.6 g SMB/kg biomass. appear. The development of surface mold spots in these silages was accompanied by a decaying odor. The amount of SMB used in silage with lower FVD rates (i.e., 2.4 and 1.6 g SMB/kg biomass) may be insufficient to inhibit the growth of perishable microorganisms and subsequent decay. Moreover, the high DM content of these silages is likely to contribute to lower compaction and efficient air exclusion, which is important for inhibiting the growth of undesirable microorganisms. Among the SMB silages, those with higher SMB loads exhibited stronger SMB odors in ensiling. However, as the storage period increases, the smell of SMB gradually decreases. This can be explained by the gradual loss of SMBs during the storage period. When the corn feed was treated with SMB (4 g SMB/kg fresh biomass) and ensailed for 90 days, the remaining bisulfite of the treated silage was about a quarter of the initial addition amount. The reason for this loss of SMB during the ensiling process is explained by the fact that metabisulfite is a strong reducing agent that is oxidized to sodium bisulfate when exposed to a reducing component. The access of air or reducing substances to bisulfite in the silage mass is expected to contribute to further lowering the residual sulfite content in the silage. Moreover, the generation of sulfur dioxide gas can be another path to SMB loss in the ensiling process.

Overall, among metabisulfite silages, a silage with a 3.2 g SMB/kg fresh mass corresponding to about 50% moisture provided the most desirable organoleptic properties. Compared to the silage with 4 g SMB/kg biomass (about 60% moisture level), the FVD ratio (53.2%) of the silage with 3.2 g SMB/kg biomass is lower, and the DM content is higher, so a more pleasant odor (Lower SMB odor), and the wastewater runoff is negligible, which can be considered more desirable.

2-5. Free sulfite concentration

Fig. 14 shows the time course of the free sulfite concentration according to the storage period in silages with various SMB throughputs. Unlike the results of Experiment 1, in which a relatively high content of free sulfite was found in the SMB silage even after 90 days of storage, the free sulfite content decreased to a negligible amount after 30 days of ensiling. Although uncertain, free sulfite appears to be readily bound to some of the constituents of almond husk or corn gluten diets. Free sulfite is the active form of SMB, which provides potent inhibition on the growth and development of microorganisms. Significant reduction in free sulfite throughput is a potential source of feed nutrients for inclusion of ensilized biomass as moist or semi-dry total blended silage (50%-65% DM), or for biogas production. It provides an opportunity for efficient use as biomass.

When mixing SMB silage as a dietary nutrient in the feed of ruminants, it is advisable to consider the sulfur content of the diet. The National Research Council (2000) reported that the sulfur requirement for feedlot livestock was 1.5 g/kg diet DM, and the maximum allowable concentration was about 4 g/kg diet. There is no loss of metabisulfite during the storage period, and the 20% dietary inclusion level of metabisulfite silage (3.2 g/kg biomass) is an additional 0.22 g/kg in the diet. Assuming conversion to sulfur, this is not practical and can be easily incorporated into a diet with some dietary transformations.

2-6. Aerobic stability

The chemical and microbiological properties of the final silage (after 90 days) exposed to air for 5 days are summarized in FIG. 17. In general, silages with lower SMB concentrations (drier silages) had significantly lower stability with respect to a substantial increase in pH and carbon dioxide production. This was accompanied by the rapid spread of yeast and mold. In contrast, silages with 4 and 3.2 g SMB/kg biomass were more stable with lower carbon dioxide production, less microbial population and less pH increase. In general, dry silage tends to be more porous than moist silage, which stimulates rapid decay when exposed to air. In general, silage with yeast exceeding 5 log cfu/g DM is expected to rapidly decay upon opening of the silage. Following oxygenation of the silage, the yeast begins to multiply rapidly, consumes a significant amount of organic acids, and the silage pH rises rapidly. This may explain the rapid decay of control silages and silages with lower SMB throughput, as these silages have a large number of yeast after 90 days of ensiling (Figure 16).

It was also confirmed that the silage with higher SMB throughput (ie 4 and 3.2 g SMB/kg biomass) is more aerobicly stable as a result of this example.

In conclusion, discarded fruits and vegetables were successfully preserved with metabisulfite after 7 days of aerobic exposure and then converted to high-moisture biomass for a long time with negligible energy costs associated with dry matter and nutrient loss. It was ensiled. The co-ensiling of FVD with almond husk and corn gluten feed, which provided an SMB throughput of 3.2 g/kg biomass, corresponding to a moisture content of about 50%, provided the best preservation properties. Overall, treating discarded fruits and vegetables with metabisulfite can be seen to be effective for short-term aerobic preservation followed by long-term anaerobic preservation without requiring fermentation of lactic acid bacteria.

[ Example 3] SMB And organic acid treatment FVD Long-term preservation

The experimental method related to long-term preservation of FVD was carried out using the same method as in Example 2, except for simultaneous treatment of SMB and organic acid.

The organic acids used were sodium benzoate, potassium sorbate, and sodium nitrite, and their mixing ratio was summarized in FIG.

After storage under aerobic conditions for 7 days, the dry weight and water-soluble carbohydrate content (WSC) content characteristics of FVD in the presence or absence of SMB and organic acids were investigated and shown in FIG. 20.

As a result, in the negative control T1, after 7 days of aerobic storage, the loss of dry matter and water-soluble carbohydrate content was significant, but both the SMB-treated test group (T2) and the organic acid-treated test group (T3) It was found that the loss of the water-soluble carbohydrate content was insignificant, and the loss of the dry matter and water-soluble carbohydrate content was also observed to be insignificant in the test groups (T4 to T6) treated with SMB and organic acid simultaneously.

In addition, microbiological and physicochemical properties of FVD in the presence or absence of SMB and organic acids after storage for 7 days under aerobic conditions are shown in FIG. 21.

Referring to FIG. 21, in the case of the negative control group, the total number of bacteria and yeast increased significantly after 7 days of aerobic storage. This means proliferation of spoilage bacteria. On the other hand, it was observed that the total number of bacteria, yeast, and fungi was lower than the detection level in the T4 and T5 treatment groups, which were test groups treated with SMB and organic acid simultaneously. This is in line with the slight changes in dry matter and water-soluble carbohydrates observed in Figure 20.

In addition, the pH characteristics of FVD in the presence or absence of SMB and organic acid after storage for 7 days in an aerobic condition are shown in FIG. 22. Referring to FIG. 22, in the case of the negative control group, a sharp decrease in silage pH was observed after 7 days of aerobic storage. On the contrary, it was observed that the pH was constantly low in the T4 and T5 treatment groups, which were test groups treated with SMB and organic acid at the same time. Similarly, these results are in line with the slight changes in dry matter and water-soluble carbohydrates observed in FIG. 20.

Through the above results, it was confirmed that the concurrent use of SMB and organic acid was effective in controlling microbial growth and preserving nutrients.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

delete delete delete Sodium metabisulfite (SMB) in fruit and vegetable discards (FVD), including grape, apple, tomato, paprika, lemon, onion, and potato waste in an aerobic environment; And a pretreatment step of simultaneously treating an acid mixture in which sodium benzoate, potassium sorbate, and sodium nitrite are mixed in a weight ratio of 4:2:1 and then storing for 6 to 9 days. And
Including an ensiling step of storing the fruit and vegetable waste subjected to the pretreatment step in a silo container, sealing it, and storing it at room temperature at 20 to 25°C for 7 to 90 days,
The sodium metabisulfite and acid mixture is treated at a rate of 2 g per 1 kg of fruit and vegetable waste (FVD), or sodium metabisulfite is 3 g per kg of fruit and vegetable waste (FVD), and the acid mixture is 1.5 g. Is processed in proportion,
In the ensiling step, almond husk and corn gluten are mixed with fruit and vegetable waste (FVD) that has undergone a pretreatment step, but the fruit and vegetable waste (FVD) is mixed in a ratio of 39.8 to 66.6% by weight, Almond husk and corn gluten comprising the step of mixing each other in the same weight percent ratio,
A method for producing feed using fruit and vegetable waste (FVD). delete delete delete delete delete delete delete delete

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