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Agricultural And Food Sciences
Medicine

text:
Research Effects of Dietary Sodium Acetate on Growth Performance, Fillet Quality, Plasma Biochemistry, and Immune Function of Juvenile Golden Pompano ( Trachinotus ovatus )

Six isonitrongenous and isolipidic diets were formulated to feed 360 ﬁ sh (mean initial body weight: 6 : 06 ± 0 : 02 g) with graded levels of sodium acetate (SA1 (0), SA2 (250mg/kg), SA3 (500mg/kg), SA4 (1000mg/kg), SA5 (2000mg/kg), and SA6 (4000mg/kg)) for 8 weeks. The results revealed that weight gain rate, speci ﬁ c growth rate, and feed intake of golden pompano were signi ﬁ cantly increased in SA3 and SA4 treatments ( p < 0 : 05 ). Dietary SA level signi ﬁ cantly improved lipid contents and gumminess of muscle ( p < 0 : 05 ). Supplement levels of dietary SA signi ﬁ cantly in ﬂ uenced plasma biochemical parameters containing triglyceride, glucose, high-density lipoprotein cholesterol, complements, and immunoglobulin M ( p < 0 : 05 ). Appropriate supplement levels of dietary SA signi ﬁ cantly increased enzyme activities of hepatic superoxide dismutase and total antioxidant capacity and decreased malondialdehyde contents ( p < 0 : 05 ). Furthermore, appropriate SA levels downregulated the expression levels of Keap1, IL-8, TNF- α , and NF- κ B genes and upregulated the expression levels of Nrf2 and TGF- β genes of the spleen ( p < 0 : 05 ). Excess SA caused negative e ﬀ ects for Trachinotus ovatus . The optimum supplement level of SA for juvenile golden pompano was evaluated to be 1423.67mg/kg based on WGR.

Introduction
The harvest of intensive aquaculture greatly relied on reducing the influence of disease in farmed fish [1]. Antibiotics has been intensively used to control disease in an aquatic organism [2]. Overused antibiotics used in feed induced hazards in health and environment, which brought about the ban of antibiotics in many countries [2]. Great attentions have arisen in seeking alternatives to antibiotics. Shortchain fatty acids (SCFAs) are thought to safe feed additives and have been applied in aquaculture recently [3]. SCFAs belong to organic acids, such as acetic acid, propionic acid, and butyric acid [4]. Dietary SCFAs exist in several forms, such as calcium, sodium, and potassium salts [5]. SCFAs are generally easily absorbed and promote digestive function of the intestinal epithelia in fish [6]. SCFAs are involved in ameliorating growth, immune responses, and intestinal microorganisms in fish [7,8].
Acetate is one of the major SCFAs and is produced from pyruvate (through acetyl-CoA or the Wood-Ljungdahl pathway), arabinoxylan (through intestinal bacteria), and several amino acids (through deamination) [3,9,10]. In aquatic animals, sodium acetate (SA) has been added in the diets of Arctic charr (Salvelinus alpinus) [11], common carp (Cyprinus carpio) [4], zebrafish [12], yellowfin seabream (Acanthopagrus latus) [6], and Siberian sturgeon (Acipenser baerii, Brandt, 1869) [13], which showed effective improvements in growth performance and immune responses. In addition, a recent study of Nile tilapia (Oreochromis niloticus) indicated that SA regulated the MAPK and NF-κB signaling pathways to alleviate high-carbohydrate-triggered intestinal inflammation [14]. The studies of dietary SA on the growth and immune functions in fish are still limited although several researches have been performed.
Golden pompano, Trachinotus ovatus is an economically important fish species because of its advantages of growth, flesh quality, and salinity tolerance [15]. Unfortunately, golden pompano often suffered from inflammation and disease due to intensive culture pattern [16]. SA supplementation might provide a new idea to relieve these symptoms of T. ovatus after the antibiotics were banned. The aim of the present study was to assess the effects of dietary sodium acetate for juvenile T. ovatus according to the growth performance, fillet quality, plasma biochemistry, and immune function.

Materials and Methods
2.1. Ethics Statement. All animal processes were approved by the Ethical Committee of Shanghai Ocean University (Shanghai, China).

Experimental
Diets. The feed formulations are presented in Table 1. Six isoproteic (442.2 g/kg) and isolipidic (106.4 g/ kg) diets were formulated through supplementing SA (0, 250, 500, 1000, 2000, and 4000 mg/kg), which were named as SA1, SA2, SA3, SA4, SA5, and SA6, respectively. SA (purity ≥ 99:0%) is provided by Sangon Biotech Co., Ltd. (Shanghai, China). Dietary protein was supplied by fish meal, soybean meal, soy protein concentrate, peanut meal, and pork powder. Dietary lipid was supplied by fish oil and soybean lecithin. All the components were pulverized into dust and sieved via a 425 μm mesh sifter. Then, they were thoroughly blended with fish oil and soybean lecithin in Hobart-type mixer (SZ25, Guangzhou Xuzhong Food Machinery Co., Ltd.) until homogenized. Next, water was supplied to shape a stiff dough and 2 and 2.5 mm diameter feed granules were obtained using a granulator. After drying, they were stored at -20°C until use.
2.3. Experimental Animals and Design. T. ovatus were purchased from an aquaculture company (Shenzhen, China). Before the beginning of trial, fish were fed SA1 diet for 14 days in polythene cages to acclimate the laboratory conditions and keep equal physiological status, followed by a 24 h of fasting. Subsequently, the experimental fish with homogenous size (average: 6:06 ± 0:02 g) were allotted in 18 pond cages (1 m × 1 m × 1:8 m) with 20 fish per cage. Fish were slowly hand-fed twice a day (7 : 00 and 17 : 00) to visible sense of satiety. The trial lasted 8 weeks and the data (number of dead fish, weight of dead fish and feed intake) in each cage were recorded daily. Water temperature was 31:06 ± 0:30°C, dissolved oxygen 7:78 ± 0:05 mg/L, salinity 15:32 ± 0:20 g/L, and pH 8:24 ± 0:08. Dissolved ammonia was ≤0.05 mg/L, and nitrite was ≤0.01 mg/L.

Sample Collection.
Before sampling, fish were fasted for 24 h. They were anesthetized with eugenol (100 mg/L) for determining the number and weight of fish per cage. Three fish per cage were sacrificed randomly for the measurement of nutrient component of carcass. Five fish per cage were sacrificed and weighed individually. Then, their blood was collected from a caudal venipuncture using 2 mL heparinized syringes. Livers, spleens, and dorsal muscles of aforementioned five fish were aseptically removed and, respectively, collected into tubes and stored at -80°C until further analysis. Another three fish in each cage were caught, and dorsal muscles were collected and immediately performed the texture characteristics analysis.
2.5. Calculations. The metrics of growth performance including the weight gain rate (WGR), specific growth rate (SGR), feed efficiency ratio (FER), feed intake (FI), condition factor (K), hepatosomatic index (HSI), viscerosomatic index (VSI), and survival rate were calculated as follows: FI g ð Þ = dry diet feed g ð Þ/number of fish, In the above equations, W f , W i , W w , W bw , W bl , W l , W v , N f , and N i are final average body weight (g), initial average body weight (g), weight gain (g), body weight of fish (g), body length of fish (cm), liver weight of fish (g), viscera weight of fish (g), finial number of fish, and initial number of fish, respectively.
2.6. Chemical Analysis. The proximate composition was measured according to the standard method of Association of Official Analytical Chemists [17]. Moisture was measured on the basis of a constant weight in oven-drying (DHG-9240, Shanghai) at 105°C. Ash was measured by a muffle furnace (FO610C, Yamato Scientific Co., Ltd., Tokyo, Japan) at 550°C for 5 h. Crude protein was determined by the acid digestion furnace (FOSS KT260, Switzerland) and an Auto Kjeldahl apparatus (Kjeldahl™ 2300 System). Crude lipid was mensurated by Soxtec™ 2055 (FOSS, Hoganos, Sweden).
Muscle samples were cut into 10 mm high cylinders before analysis. Texture characteristic measurements were performed using a texture analyzer (TC3, Brookfield, USA) and were estimated based on texture profile analysis (TPA) following a previous method [18]. The model of spherical probe was TA44, and the displacement was 3 mm. The test speed was 0.1 cm/s, and the trigger values were 5 g (m/s 2 ).

RNA Extraction and Gene Expression.
Total RNA was isolated from the spleen tissue of T. ovatus adopting the HiPure University RNA Mini Kit (Magen, Guangzhou, China) according to the manufacturer's instructions and dis-solved in DEPC treat water. Then, the quality of total RNA was determined by 1.2% agarose gel electrophoresis, and the concentration of total RNA was detected using Nano-Drop 2000 (Thermo Scientific, USA). The absorbance ratios at 260 nm and 280 nm (260/280) of all RNA samples were ranged from 1.8 to 2. The synthesize of complementary DNA (cDNA) was performed using Evo M-MLV RT Kit with gDNA Rraser (AG, Guangzhou, China) and stored at -80°C. The specific primer pairs are listed in Table 2. qPCR determination was conducted as described our previous methods [20]. All samples were determined in triplicate. Relative mRNA levels of target genes were calculated by the 2 -ΔΔCt method [21].

Statistical
Analysis. Experimental data were presented as means ± standard error of mean ðSEMÞ. The normality and homogeneity of variance were confirmed using the Kolmogorov-Smirnov test and Levene's test before performing the one-way analysis of variance (ANOVA). Then, Tukey's test was conducted to assess differences in the treatments through SPSS 23.0 (SPSS, Michigan Avenue, Chicago, IL, USA). The level of statistical significance was set at p < 0:05. A principal component test was conducted to describe the response of dependent variable to dietary SA levels via Table 1: Formulation and nutrition levels of the diets (g/kg dry weight).

Results
3.1. Growth Performance. The effects of supplement levels of dietary SA on growth performance are shown in Table 3. FBW, WGR, SGR, and FI of T. ovatus fed SA3 and SA4 diets were significantly higher than those fed SA1 diets (p < 0:05). Fish fed SA1, SA2, SA3, SA4, and SA5 showed significantly higher FBW, WGR, SGR, and FI than those fed SA6 diets (p < 0:05). The peaks of FBW, WGR, SGR, and FI were recorded for SA4 treatments. There were no significant differences in FER, VSI, HSI, K, and survival among all groups (p > 0:05). The optimal supplement level of SA was assessed to be 1423.67 mg/kg by a quadratic regression model based on WGR (Figure 1). PCA biplot and loadings showed that strong interdependency between the dietary SA level and dependent variables (FBW, WGR, SGR, and FI), with correlation as 0.9501, 0.9177, 0.9089, and 0.9346, respectively ( Figure 2).

Nutrient Component of Carcass and Dorsal Muscle.
The results are presented in Table 4. Supplement levels of dietary SA did not significantly affect whole-body composition of juvenile T. ovatus. Crude lipid of dorsal muscle in the SA4 group was significantly higher than that in SA1 treatment (p < 0:05). The differences in crude protein, ash, and moisture among all groups were not significant.
3.3. Texture Characteristics of Dorsal Muscle. The results are shown in Table 5. The texture parameters containing hardness, elasticity, chewiness, and adhesiveness did not have statistical differences among the six treatments (p > 0:05). Gumminess of dorsal muscle in the SA3, SA4, and SA5 treatments was significantly higher than that in SA1 treatment (p < 0:05).

Plasma Biochemistry and Immune
Response. As shown in Table 6, the biochemistry and immune indices of plasma were significantly affected by supplement levels of dietary SA. TP and HDL-c content in plasma of juvenile golden pompano fed SA-supplemented diets (except for SA2 diets) were significantly higher than those fed SA1 diets (p < 0:05). The highest TP and HDL-c were recorded for the SA4 and SA6 groups, respectively. Similarly, fish fed SA-supplemented diets (except for SA2) had significantly lower TG and GLU compared to that fed SA1 diets (p < 0:05). AST activities of juvenile golden pompano fed SA2, SA3, SA4, and SA5 diets were significantly lower than those fed SA1 diets (p < 0:05). The SA4 group had significantly lower ALT activities compared to the SA1 group (p < 0:05). Fish fed SA3, SA4, and SA5 diets had significantly higher C3 and IgM than the group fed SA1 diets (p < 0:05). The highest contents of C3 and IgM were recorded for the SA4 and SA3 groups, respectively. C4 contents in the SA2, SA3, and SA4 groups were significantly higher than that in SA1 (p < 0:05). There was no significant difference in plasma TC among the groups (p > 0:05). PCA presented a strong relationship between dietary SA level and dependent variables (C3, C4, AST, and ALT), and the correlation was, respectively, 0.8735, 0.8385, 0.9283, and 0.8959 (Figure 2).

Antioxidant Capacity in Liver.
Supplement levels of dietary SA significantly influenced the antioxidant capacity of liver ( Table 7). The activities of SOD in SA2, SA3, and SA4 treatments were significantly higher than that in SA1 treatment (p < 0:05). Fish fed SA4 diets had significantly higher T-AOC compared to that fed SA1 diets (p < 0:05). SAsupplemented diets significantly decreased MDA contents (p < 0:05). POD activities were not significantly influenced by supplement level of SA (p > 0:05). PCA showed a relationship between dietary SA level and dependent variables (SOD, T-AOC and MDA), with correlation as 0.8165, 0.6095, and 0.6710, respectively (Figure 2).  Figure 3. Fish fed SA-supplemented diets had significantly lower expression levels of Il-8, TNF-α, NF-κB, and Keap1 genes than that fed SA1 diets (p < 0:05).
The lowest mRNA level of IL-8 was recorded in SA3 while the lowest expression levels of Keap1, TNF-α, and NF-κB were recorded in SA4. The expression levels of TGF-β genes in SA3 and SA4 were significantly higher than that in SA1 (p < .05). The expression levels of Nrf2 genes of juvenile golden pompano fed SA2, SA3, and SA4 diets were significantly higher than that fed SA1 diets (p < 0:05).

Discussion
Administration of sodium acetate exerts positive effects on growth performance in fish [11,22]. Acetate also plays a beneficial role in appetite regulation [23]. Studies on zebrafish displayed that SA promoted appetite and FI of zebrafish [12,24]. Similarly, this study showed appropriate supplement levels of SA significantly promoted WGR and FI of juvenile golden pompano. These findings inferred that moderate SA in diets increased FI through an improvement in appetite, thus promoting growth of juvenile T. ovatus. In the current study, the optimal level of dietary SA was evaluated to be 1423.67 mg/kg based on a quadratic regression model of WGR, which was similar to the previous studies on Arctic charr (Salvelinus alpinus) [11], zebrafish [12] and Nile tilapia (Oreochromis niloticus) [14]. Hardness, elasticity, chewiness, adhesiveness, and gumminess are major indices to assess fillet quality [25,26]. Decreased lipid content in muscle will enhance the friction between muscle bundle and reduce muscle tenderness [27] as well as decreasing sensory scores for juiciness [28]. In this study, lipid content of muscle significantly increased when fish fed SA4 diets. Furthermore, gumminess of muscle increased significantly in SA3, SA4 and SA5 treatments  5 Aquaculture Nutrition compared to that in SA1 treatment. A recent study showed muscle lipid content of fish fed SA diets had an increasing trend although this was not significant [29]. These results suggested moderate SA induced muscle lipid accumulation and improved fillet quality of juvenile golden pompano to a certain extent.   Note: different superscripts within rows indicate significant differences (p < 0:05).

Aquaculture Nutrition
Health status and growth of fish go hand in hand in ensuring maximum outputs during harvest [1]. Biochemical indices of plasma can evaluate the healthy status of fish tissues [19]. TP including albumin and globulin can reflect immune status of organisms [30]. HDL-c is involved in the transport of cholesterol [19]. In the present study, SAsupplemented diets (except for SA2) significantly increased TP and HDL-c contents of fish and significantly decreased TG and GLU levels of fish. These results indicated adding moderate SA in diets had positive effects on hypolipidemic and hypoglycemic for juvenile golden pompano. AST and ALT are considered useful indices to estimate the status of liver damage [31]. Generally, increase of AST and ALT levels indicate dysfunction in liver [19]. In this study, AST and ALT contents first decreased and then increased with the incremental levels of dietary SA, which suggested appropriate SA levels heightened the protection for liver function of Trachinotus ovatus.
SCFAs can cause beneficial effects on immune response and inflammation process [32]. Many studies have confirmed vital effects of C3, C4, and IgM on immune system in fish ( [19,33,34]). A recent study reported butyrate increased complement and IgM levels as well as upregulating the mRNA level of immune-related genes [7]. The study of common carp revealed that alkaline phosphatase and total immunoglobulin contents of skin mucus remarkably increased when fish fed SA-supplemented diets [4]. In the present study, dietary SA level significantly increased C3, C4, and IgM contents, which indicated that appropriate supplement levels of SA enhanced immune response of golden pompano. The immune system and antioxidant system go hand in hand. Oxidative stress means the dynamic imbalance of redox in organisms, and it can cause overproduction of reactive oxygen species (ROS) and decrease of antioxidative enzyme activities as well as contributing to oxidative damage [35]. Oxidative stress is regarded as an important factor leading to disease and death in aquatic animals [36]. In fish, the antioxidant systems include nonenzymatic compounds and antioxidant enzymes (SOD, T-AOC, POD, glutathione reductase, and glutathione peroxidase), which play crucial roles in resisting oxidative stress [37]. SOD can decrease the level of ROS via catalyzing the highly reactive superoxide radical to H 2 O 2 [19]. POD is deemed to participate in eliminating H 2 O 2 , and T-AOC is used to assess antioxidant ability of fish [19,38]. MDA is a major production of lipid peroxidation, and its content can evaluate the status of lipid peroxidation and cell injury in organisms [39]. In this study, SA-supplemented diets can effectively increase hepatic SOD and T-AOC activities and decrease hepatic MDA contents of golden pompano. These findings indicated that moderate SA levels in diets enhanced antioxidative ability of golden pompano. A study on common carp also showed SA diets significantly upregulated the expression of genes related to antioxidant defence such as glutathione S-   [4]. Similarly, sodium propionate upregulated the expression of SOD and CAT genes in zebrafish [2]. The spleen of fish is a vital organ, which plays an important role in immunity [40]. Our study showed that appropriate supplement levels of dietary SA significantly downregulated expression of Keap1 gene and upregulated expression of Nrf2 gene in spleen of golden pompano. Nrf2, a key transcription factor involved in the combining to antioxidant response elements and regulating the expressions of antioxidative enzyme genes, can protect organisms from oxidative stress [41]. Keap1, serving as a Nrf2-binding protein, can suppress expression of Nrf2 to  Figure 3: Relative expression of immune-related genes in spleen of juvenile golden pompano fed diets containing graded levels of sodium acetate (SA). Different superscript letters above the bar represent significant differences between treatments (p < .05). 8 Aquaculture Nutrition attenuate cells antioxidative ability [42]. The results implied that moderate supplement levels of dietary SA promoted antioxidative ability and reduced the oxidative stress of golden pompano. Oxidative stress can cause inflammation and vice versa. Inflammation is an essential organismal defensive mechanism which was induced by chemical, physical, and biotic stimuli [43]. Recent studies found SCFAs involved in regulation of proinflammatory and antiinflammatory mediators through multiple ways. Sodium butyrate diets stimulated downregulation of the intestinal proinflammatory cytokines (such as IL-1β) and upregulation of the intestinal anti-inflammatory (such as IL-10 and TGF-β) in Pengze crucian carp (Carassius auratus Pengze) [7]. Administration of microencapsulated sodium butyrate in common carp is shown to increase the immunoreaction by regulating the expressions of gut proinflammatory and anti-inflammatory cytokines [44]. In the research of common carp, the mRNA levels of immune-related genes in the intestine had an upward trend when fish fed SA diets [4]. In the current study, appropriate levels of SA significantly increased expression of anti-inflammatory TGF-β cytokine and decreased expressions of proinflammatory IL-8 and TNF-α cytokines. The NF-κB signaling pathway participates in innate and adaptive immune responses as well as regulating inflammatory cytokines [41]. Recent studies showed the anti-inflammatory mechanism of SCFAs was associated with the NF-κB pathway [14,45]. Diets containing 200 mmol/L SA significantly suppressed intestinal NF-κBp65 phosphorylation on Nile Tilapia [14]. Consistent with previous study, our results showed that a significant downregulation was observed on expressions of splenic NF-κB when fish fed SA-supplemented diets. These findings indicated that dietary SA relieved inflammation and enhanced immune response of juvenile golden pompano. Interestingly, when supplement levels of SA reached SA5 (2000 mg/kg) or SA6 (4000 mg/kg), the growth performance, fillet quality, and immune function presented adverse changes for the health of golden pompano. It has been reported that the administration of exogenous acidifying agents such as acetic acid may cause disorders of acid-base balance and metabolic acidosis [46], which inferred that excessive SA would cause a toxic effect and damage physiological function of juvenile golden pompano. A previous study reported that excessive sodium butyrate decreased the growth performance of juvenile turbot [47], which supported our study. However, dietary SA did not result in the adverse changes in the studies of Arctic charr (Salvelinus alpinus) [11] and Nile tilapia (Oreochromis niloticus) [14]. This may be because the maximum supplement level of SA in these studies was lower than that in our study, which did not reach the threshold for toxicity. The specific mechanism still needs to be determined.

Conclusions
Appropriate supplement levels of dietary SA promoted growth and fillet quality of juvenile Trachinotus ovatus. The optimum supplement level of SA was explored to be 1423.67 mg/kg for juvenile golden pompano based on a qua-dratic regression model of WGR. Additionally, moderate supplement levels of SA improved antioxidative ability and immune response as well as decreasing plasmatic glucose and lipid contents of juvenile golden pompano. Excess SA in diets inhibited the growth and reduced the immune function of juvenile Trachinotus ovatus.

Data Availability
Data were available from the corresponding authors by reasonable request.

Conflicts of Interest
All authors declare no conflict of interest.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

text:
In Vivo Digestibility of Carbohydrate Rich in Isomaltomegalosaccharide Produced from Starch by Dextrin Dextranase

Slowly digestible carbohydrates are needed for nutritional support in diabetic patients with malnutrition. They are a good source of energy and have the advantage that their consumption produces a low postprandial peak in blood glucose levels because they are slowly and completely digested in the small intestine. A high-amount isomaltomegalosaccharide containing carbohydrate (H-IMS), made from starch by dextrin dextranase, is a mixture of glucose polymers which has a continuous linear structure of α-1,6-glucosidic bonds and a small number of α-1,4-glucosidic bonds at the reducing ends. It has a broad degree of polymerization (DP) distribution with glucans of DP 10－30 as the major component. In our previous study, H-IMS has been shown to exhibit slow digestibility in vitro and not to raise postprandial blood glucose to such levels as that raised by dextrin in vivo. This marks it out as a potentially useful slowly digestible carbohydrate, and this study aimed to evaluate its in vivo digestibility. The amount of breath hydrogen emitted following oral administration of H-IMS was measured to determine whether any indigestible fraction passed through to and was fermented in the large intestine. Total carbohydrate in the feces was also measured. H-IMS, like glucose and dextrin, did not result in breath hydrogen excretion. Carbohydrate excretion with dietary H-IMS was no different from that of glucose or water. These results show that the H-IMS is completely digested and absorbed in the small intestine, indicating its potential as a slowly digestible carbohydrate in the diet of diabetic patients.

INTRODUCTION
The number of older patients with diabetes is increasing in developed countries. 1) As these patients are at risk of malnutrition and sarcopenia, it is recommended that they maintain adequate energy intake while maintaining control of blood glucose levels. 2) Oral nutrition supplements are a useful way to improve malnutrition in these patients. These typically consist of liquid diets containing the starch degradation product dextrin as a carbohydrate source. As dextrin is rapidly digested and absorbed in the human digestive tract, it is a useful energy source for the older people with malnutrition, but it tends to cause high blood glucose levels after ingestion in diabetics. Carbohydrates that are digested more slowly, but still completely, produce low blood glucose peaks and would be ideal for older patients with diabetes.
Such carbohydrates are called slowly digestible carbohydrates (SDCs). 3)4) SDCs have mild resistance to digestive enzymes in the gastrointestinal tract and are slowly absorbed in the small intestine. Nevertheless, they are eventually completely absorbed in the small intestine and are expended as energy in the body (approximately 4 kcal/g) at a level similar to that of rapidly digestible carbohydrates (RDCs) such as dextrin and starch. 5) Carbohydrates that have strong resistance to digestive enzymes are termed indigestible carbohydrates (IDCs). IDCs pass through the small intestine and are fermented by intestinal bacteria in the large intestine or excreted in the feces. 5)6) Although IDCs have nutritional roles, acting as dietary fiber and prebiotics, they have lower energy value than RDCs and SDCs. 7) In a previous study, we developed a new carbohydrate material by treating starch hydrolysate with dextrin dextranase (DDase), α-amylase, isoamylase, and pullulanase. 8) This material is a mixture of glucose polymers whose degree of polymerization (DP) covers a wide range. The main structural component of the glucans is α-1,6-glucan, which has a continuous linear structure of α-1,6-glucosidic bonds and a small number of α-1,4-glucosidic bonds at the reducing ends. Since carbohydrates with size range between DP 10 and 100 (or 200) are defined as megalosaccharides, 9) α-1,6-glucans in this DP range are isomaltomegalosaccharides (IMS). 10) In the newly developed material, about half of it were α-glucans in the range of DP 10 to 150, and the main component was α-1,6-glucan in DP 10 to 30. 8) Therefore, the material is high-amount IMS containing carbohydrate (H-IMS). The basic structure of H-IMS is similar to that of low-molecular-weight dextran, except that it has α-1,4-bonds at the reducing end. In addition, we reported that H-IMS has a slower digestion rate in vitro and produces a lower increase in blood glucose in rats than dextrin. However, it is unclear whether the result is because the H-IMS contains IDCs or because it contains SDCs that are slowly and completely digested and absorbed. Dextran, a very long-chain α-1,6-glucan, is known to be indigestible, 11) and isomaltooligosaccharide, an α-1,6-glucan (DP 2 to 5), is known to be completely digested in the small intestine. 12) However, there have been no studies investigating the in vivo digestibility of an α-1,6-glucan whose DP is intermediate between dextran and isomalto-oligosaccharide. The purpose of this study was to evaluate the digestibility of H-IMS in the small intestine to determine whether it could be classified as a SDC.

Preparation of H-IMS.
DDase was prepared from the culture supernatant of Gluconobacter oxydans ATCC 11894 by methods previously reported. 8) One unit of DDase enzyme activity was defined as the amount of the enzyme producing 1 µmol of maltotriose per min in a reaction solution containing 0.5 mL of DDase solution and 0.5 mL of 2 % (w/v) maltotetraose dissolved in 50 mM acetate buffer (pH 4.2) kept at 35 C. H-IMS was prepared as previously described, 8) with some modifications. Corn starch was industrially hydrolyzed by the general liquefaction method using α-amylase in the factory (Nihon Shokuhin Kako Co., Ltd., Shizuoka, Japan). Its dextrose equivalent value, as estimated by the Somogyi modified method, was 6.5. DDase (5 units/g substrate), α-amylase (0.1 units/g substrate, Kleistase L-1; Amano Enzyme Co., Ltd., Aichi, Japan), isoamylase (200 units/g substrate, GODO-FIA; GODO SHUSEI CO., LTD., Tokyo, Japan), and pullulanase (0.6 units/g substrate, Pullulanase Amano 3; Amano Enzyme Co., Ltd.) were added to 24 % (w/w) starch hydrolysate solution. The enzyme activity values for α-amylase, isoamylase, and pullulanase were as reported by their manufacturers. The reaction solution was kept at 50 C, pH 5.0 for 120 h. The reaction was stopped by adjusting the pH to 4.0 and the mixture was then maintained at 80 C for 12 h. The obtained product was desalted by an ion exchange resin (Amberlite MB4, Organo Corporation, Tokyo, Japan), filtered, and lyophilized. The DP distribution of the product was determined by high-performance liquid chromatography as follows: two OHpak SB-806M HQ columns (8.0 300 mm; Showa Denko K.K., Tokyo, Japan) connected in series; column temperature, 50 C; flow rate, 0.3 mL/min; eluent, 50 mM sodium nitrate solution; detector, refractive index detector. A pullulan standard (Shodex Standard P-82, Showa Denko K. K.) was used to detect the elution time. The content of DP 1 9, DP 10 30, DP 31 150, and DP 151 or higher, which was judged based on the elution time, was 36, 48, 8, and 8 %, respectively ( Fig. 1). Glycosidic bond formulation of the product was calculated from the peak area in 1 H-NMR. NMR was measured with a Bruker 400 MHz NMR spectrometer (Bruker Japan K.K., Kanagawa, Japan) at room temperature in deuterated water. Proportion of linkage was determined from the area ratio of the H-1 peaks at 4.9 ppm for α-1,6-glucosidic bonds and 5.3 ppm for α-1,4-glucosidic bonds. The proportion of α-1,4 linkage and α-1,6 linkage was 24 and 76 %, respectively. This proportion is similar to IMS prepared by Lang et al. 10) rather than dextran (molecular mass, 1,270 kDa) prepared by using DDase. 13) In vitro digestibility. The in vitro digestibility of the H-IMS was evaluated as previously described. 8) Briefly, rat small intestine acetone powder (Sigma-Aldrich Japan Co., Llc., Tokyo, Japan) was suspended in 45 mM maleate buffer (pH 6.6) at a concentration of 10 % (w/v) and centrifuged at 19,000 G for 10 min. One unit of activity of the supernatant (rat intestinal crude enzyme) was defined as the amount of the enzyme hydrolyzing 1 µmol of maltose per min at 37 C in a reaction solution containing 15 µL of the rat intestinal crude enzyme and 85 µL of 1.1 % (w/v) maltose dissolved in 45 mM acetate buffer (pH 6.0). Dextrin (Pinedex#2, Matsutani Chemical Industry Co., Ltd., Hyogo, Japan) or H-IMS was dissolved to a final concentration of 0.45 % (w/v) in 45 mM maleate buffer (pH 6.6). Porcine pancreatic α-amylase (Type I-A, Sigma-Aldrich Japan) and the rat intestinal crude enzyme were added to the substrate solution at a concentration of 4,000 and 860 units/g substrate, respectively, and the solution was kept at 37 C. One unit of activity of α-amylase was defined as the amount of the enzyme producing 1 mg of maltose per 3 min at 20 C and pH 6.9. Then, 200 µL of 2 M Tris-HCl solution was added to 20 µL of the reaction solution to stop the reaction of rat α-glucosidase at each reaction period. The glucose concentration was measured by the glucose oxidase method using the Glucose C-II Test (Wako Pure Chemical Industries, Osaka, Japan). In vivo experiments. Animals were treated in accordance  Postprandial breath hydrogen gas. The measurement of breath hydrogen gas as an indicator of fermentation in the large intestine was performed according to previously published methods, 15) with some modification. From two days prior to the test day, rats (n = 24, 8 weeks old) were housed on wire-mesh flooring and fed the gas-free diet. On the test day, each animal was assigned to one of four treatment groups: glucose, dextrin, lactulose, or H-IMS (n = 6 each). Chambers with two air vents and a capacity of 3.4 L (air-tight chamber, Muromachi Kikai Co., Ltd., Tokyo, Japan) were aspirated continuously at a flow rate of 200 mL/ min from one vent with a pump connected to a tube (Pomp: Masterflex L/S, Tube: Noprene Tubing, Cole-Parmer Instrument Co., Ltd., Illinois, USA), while fresh air was allowed to enter from the other vent. For breath collection at 0 h, each rat was placed in the chamber for 20 min and then aspirated internal air was collected through the tip of the tube for 1 min into a bag (Collection bag, Laboratory for Expiration Biochemistry Nourishment Metabolism Co., Ltd., Nara, Japan). After the collection at 0 h, glucose (Nihon Shokuhin Kako Co., Ltd.), dextrin (TK-16, Matsutani Chemical Industry Co., Ltd.), lactulose (MLC-97, Morinaga Milk Industry Co., Ltd., Tokyo, Japan), or H-IMS was dissolved in distilled water well in advance at a concentration of 0.2 g/ mL and was orally administered at a dose of 2 g/kg body weight (10 mL/kg body weight). Each rat was placed in the chamber for 20 min every hour until 9 h after administration and aspirated air samples were collected as described above. Feed was removed from the start of the 0-h collection to the end of 9-h collection. The hydrogen concentration in the bag was measured with a Breath Gas Analyzer (BGA-2000D, Laboratory for Expiration Biochemistry Nourishment Metabolism Co., Ltd.). Excretion in stool. The measurement of fecal excretion of total carbohydrate was performed according to methods previously described, 16) with some modifications. From one week prior until four days after oral administration, rats (n = 24, 7 weeks old) were housed on wire-mesh flooring and fed the starch-free diet. On treatment day, each animal was assigned to one of four groups: water, glucose, polydextrose, or H-IMS (n = 6 each). For the water group, water was administrated at 10 mL/kg body weight. For the other three groups, glucose, polydextrose (Litesse, Danisco Japan Co., Ltd., Tokyo, Japan), or H-IMS was dissolved in distilled water at a concentration of 0.2 g/mL and administrated orally at a dose of 2 g/kg body weight (10 mL/kg body weight). Stool was collected and weighed every day for 4 days. Total fecal wet weight and food intake during these 4 days were also measured. The amount of total carbohydrate in the stool samples was determined by the phenol-sulfuric acid method, 17) with glucose used as the standard. The measured value quantified as glucose equivalent was converted to amount of polysaccharide by multiplying by a polysaccharide coefficient of 0.9. Fecal excretion rate for each animal was calculated by first subtracting the mean fecal carbohydrate excretion in the water group from the fecal carbohydrate excretion in the animal followed by dividing by the oral dose in each rat. Statistical analysis. Data are reported as mean standard error of the mean (SEM). Statistical analyses were performed using JMP statistical software (version 13.2.1, SAS Institute Japan Inc., Tokyo, Japan). The non-parametric Steel-Dwass test was used for multiple comparisons of the differences at each time point in the breath hydrogen tests, as these values did not show a normal distribution. The Tukey-Kramer test was used for multiple comparisons in the fecal excretion test. Differences at p < 0.05 were considered significant.

In vitro digestibility of H-IMS.
Dextrin was rapidly digested after the addition of the enzyme solution containing α-amylase and rat small intestine mucosa, with released glucose reaching saturation after 4 h (Fig. 2). In contrast, H-IMS was digested more slowly, with released glucose levels relative to dextrin after

Breath hydrogen gas.
To investigate the fermentation of carbohydrates in the large intestine, we assessed the amount of hydrogen present in exhaled breath following treatment. Figure 3 shows the postprandial hydrogen concentration in the test chamber after oral administration of glucose, dextrin, H-IMS, or lactulose. With lactulose, hydrogen was detected from 2 h after administration, and the concentration continued to increase during the observation period, up to 9 h after administration. No increase in hydrogen concentration was observed after administration of H-IMS, glucose, or dextrin.

Fecal carbohydrate excretion.
The total amount of carbohydrate contained in the stool on the 4 days following administration of water, glucose, H-IMS, or polydextrose is shown in Fig. 4. Fecal carbohydrate excretion in the polydextrose group peaked on the first day after administration and then continued to decrease, only reaching a level not significantly different from that of the water group on day 4. In contrast, the amount of fecal carbohydrate excreted in the H-IMS, glucose, and water groups remained at the same low level for all 4 days, with no significant differences in values among them.
A summary of this study s results is shown in Table 2. There was no significant difference in total food intake or in total wet weight of stool between groups for the 4 days after treatment. However, total carbohydrate excretion was about seven times higher in the polydextrose group than in the other three groups. Incremental value of carbohydrate excretion in the polydextrose group was 199.9 17.5 mg/4 days, an amount equivalent to 35.1 % of the mean oral dose. In contrast, the values for total amount of carbohydrate excreted in feces in the H-IMS and glucose groups were equivalent to those in the water group, indicating no increase in fecal carbohydrate amount due to the administration of these carbohydrates.

DISCUSSION
Earlier, we reported that H-IMS has a slower in vitro digestion rate and produces a lower increase in blood glucose in rats than dextrin. 8) In this report, we evaluate the in vivo digestibility of H-IMS in rats. We have found that H-IMS is completely digested and absorbed in the small intestine. To the best of our knowledge, this is the first report on the in vivo digestibility of an α-1,6-glucan mixture containing IMS as the major component.
Hydrogen gas is generated when carbohydrates escape digestion in the small intestine and reach the large intestine, where they are fermented by the intestinal anaerobic bacteria. 18)19) This hydrogen diffuses into the blood and is then detectable in exhaled breath. Since approximately 70 % of gut bacteria are capable of producing hydrogen, detection of exhaled hydrogen is an effective indicator of carbohydrate fermentation in the large intestine. 20)21) It has been reported that treatment with lactulose, an IDC, results in breath hydrogen levels of 10 and 20 ppm at 4 and 24 h post-treatment, respectively, at a dose of 2 g/kg body weight in rats. 15) In this study, lactulose exhibited exhaled hydrogen at same level as in the previous study (Fig. 3). The previous study also reported that both digestible sucrose (known to be a RDC) and isomaltulose (known to be a SDC 22) ) did not produce an increase in exhaled hydrogen after administration in rats. 15) In this study, measurements were completed up to 9 h after the animals were fed with H-IMS. Detectability of breath hydrogen of fermentable IDC usually starts with a delay of 2 to 4 h after feeding and lasts for many hours. 15)23)24) Thus, we considered, breath hydrogen should have been detected by 9 h after DDase ingestion at the latest. In the absence of detectable breath hydrogen, we conclude that fermentation in the large intestine does not occur after ingestion of H-IMS.
Next, to determine whether the H-IMS constituted a biologically unavailable component that is excreted in feces without being absorbed in the small intestine or fermented by intestinal bacteria, we evaluated the fecal carbohydrate excretion after administration. By the same method, a previous study reported that indigestible maltodextrin had a fecal excretion rate of 38 %, which is the basis for setting the energy value of the product. 16) That study also showed that the fecal excretion rate of dextrin was 0 %, indicating that digestible carbohydrates were not excreted in the feces. We used polydextrose as a positive control because it is soluble and has a relatively high (about 50 %) fecal excretion rate, as reported in previous studies using 14 C-labeled polydextrose. 6)25) 26) In our experiments, the average 4-day fecal excretion rate of polydextrose was lower, about 35 % of the oral dose ( Table 2). The difference may be due to a difference in measurement protocol: in the previous studies 14 C-radioactivity from orally administered 14 C-polydextrose was detected, and whole stool samples were used without centrifugation. 6)25)26) Under those circumstances, radioactivity readings could have detected by 14 C-containing metabolites produced and excreted or retained by intestinal bacteria in addition to polydextrose itself, because polydextrose has partial fermentability in the large intestine. 26) In contrast, we restricted the detection to carbohydrates in the supernatant of the stool, which may explain the lower apparent excretion rate of polydextrose. However, the excretion of H-IMS could be determined by detecting the carbohydrates (excreted H-IMS or its degradation product) in the fecal supernatant, because it has no fermentability as determined by the breath hydrogen test. About 70 % of the total excretion of polydextrose occurred on the first day after administration, and by the fourth day, the difference in excretion level from the water-administered group was negligible (Fig. 4). Therefore, it was considered necessary and sufficient to collect stool for 4 days after administration to evaluate carbohydrate excretion in stool. Our result shows that H-IMS has almost no unavailable component that would pass through to the large intestine and be excreted from the stool. Therefore, we consider that H-IMS is a carbohydrate that is completely digested and absorbed in the small intestine because neither fermentation in the large intestine nor excretion into the feces was clearly observed.
The α-1,6-glucosidic linkages in dietary carbohydrates are decomposed at a slower rate than the α-1,4 linkages by the α-1,6-glycosidic bond hydrolysis activity of sucraseisomaltase in the small intestinal mucosa, releasing glucose from the non-reducing end. 27) Therefore, the digestibility of α-1,6-glucans is considered to be affected by their DP. In fact, the higher the DP of samples, the lower the in vitro digestion rate, as we learned in the previous study in which we prepared α-1,6-glucan samples adjusted to various DP ranges by hydrolysis and fractionation of dextran. 8) We speculate that α-1,6-glucans with DP below a certain level, such as the H-IMS, low-molecular-weight dextran in the same DP range as the H-IMS, as well as isomalto-oligosaccharide, 12) are completely digested in the small intestine. On the other hand, α-1,6-glucans with a high DP, such as high-molecular-weight dextran, are considered to be partially indigestible in vivo because they need too long time to be digested completely. 11) Generalization of our findings to human alimentation of H-IMS is not straightforward because we did not assess dose dependency. We evaluated in vivo digestibility by a single administration of H-IMS at a dose of 2 g/kg body weight, which was used in the previous study about digestibility of isomaltulose. 15) Since our goal is to use H-IMS as a nutrition source, further investigations will be needed to evaluate its digestibility at high doses.
The animal model rather than humans are used because H-IMS is a newly developed carbohydrate. Previous investigations of the digestibility of other carbohydrates reported almost the same postprandial breath hydrogen pattern of resistant glucan 23) and fecal excretion rate of resistant maltdextrin 16)28) in rats as in humans. These findings suggest that the rat is a good animal model for human carbohydrate digestion and its use is an appropriate first step prior to human trials. In the future, to estimate the digestibility and energy value of the H-IMS in humans, breath hydrogen measurement, 12) indirect calorimetry, 28)29) and evaluation of breath carbon dioxide excretion using a tracer 6)15) in humans would be needed.
This study suggests that the H-IMS is a SDC that would be useful for energy supplementation in diabetic patients. Although isomaltulose 22) and trehalose 30) have already been developed as SDCs, they are disaccharides, and isomaltulose contains fructose as one of its two constituent sugars. In contrast, the H-IMS is a polysaccharide constructed only from glucose, as well as starch. Disaccharides tend to cause increased osmotic pressure in the gastrointestinal tract, which leads to diarrhea, 31)32) The effects of chronic fructose intake on metabolism are still controversial. 33) Considering daily intake, the advantage of the H-IMS is that its structure is similar to that of starch, the main nutrient source for humans, and it can be produced from starch, an inexpensive raw material.

CONCLUSIONS
The H-IMS, which is a linear α-1,6-glucan-based carbohydrate produced using DDase, showed neither generation of breath hydrogen nor increased fecal sugar excretion after administration in vivo; therefore, it is considered that the H-IMS is almost completely digested and absorbed in the small intestine. The H-IMS could be a beneficial SDC as the nutrition source that provide energy while avoiding rapid postprandial blood glucose increase in diabetic patients.


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A Green Extraction Method to Achieve the Highest Yield of Limonin and Hesperidin from Lime Peel Powder (Citrus aurantifolia)

Green extraction is aimed at reducing energy consumption by using renewable plant sources and environmentally friendly bio-solvents. Lime (Citrus aurantifolia) is a rich source of flavonoids (e.g., hesperidin) and limonoids (e.g., limonin). Manufacturing of lime products (e.g., lime juice) yields a considerable amount of lime peel as food waste that should be comprehensively exploited. The aim of this study was to develop a green and simple extraction method to acquire the highest yield of both limonin and hesperidin from the lime peel. The study method included ethanolic-aqueous extraction and variable factors, i.e., ethanol concentrations, pH values of solvent, and extraction temperature. The response surface methodology was used to optimize extraction conditions. The concentrations of limonin and hesperidin were determined by using UHPLC-MS/MS. Results showed that the yields of limonin and hesperidin significantly depended on ethanol concentrations and extraction temperature, while pH value had the least effect. The optimal extraction condition with the highest amounts of limonin and hesperidin was 80% ethanol at pH 7, 50 °C, which yields 2.072 and 3.353 mg/g of limonin and hesperidin, respectively. This study illustrates a green extraction process using food waste, e.g., lime peel, as an energy-saving source and ethanol as a bio-solvent to achieve the highest amount of double bioactive compounds.

Introduction
Plant extract is one of the main ingredients in functional food, dietary supplements and herbal medicine. Production of plant extract usually requires growing a large amount of plant as raw materials and utilizing toxic solvents for extraction. Thus, a green extraction concept has been proposed as a desirable approach to save energy, reduce environmental problems, and promote sustainable development [1]. Chemat et al. proposed that "green extraction is based on the discovery and design of extraction processes which will reduce energy consumption, allow the use of alternative solvents and renewable natural products, and ensure a safe and high-quality extract/product" [1]. To obtain limitless and energy-saving plant sources for extraction, clean waste from the food industry can be a good candidate.
Lime (Citrus aurantifolia) is a citrus fruit that has been cultivated around the world. Its annual world production is approximately 20,050,000 metric tons [2]. The fruit is an industrial drop that is sold in both fresh markets and the food industry. Nevertheless, the commonly consumed part of the lime is the juice, which accounts for about 48% of the total weight, while the rest (peel, seed, and dehydrated flesh: 52% of total weight) is usually unused, thus becoming food waste [3]. To promote responsible consumption and production, according to the sustainable development goals, such a significant amount of food waste from lime use should be managed and comprehensively exploited. Utilizing food waste such as the lime peel not only reduces environmental problems but also adds commercial value to limes [4].
Previous studies have shown that lime is a rich source of phytochemical compounds, especially flavonoid and limonoid groups. These compounds can be found in all parts of the lime, mostly abundant in the outer and inner layers of the peel (flavedo and albedo) [3,5,6]. The prominent phytochemical compounds that are reported to be found in waste products from lime are limonin and hesperidin. Limonin is a natural tetracyclic triterpenoid compound in the limonoid group. It is a secondary metabolite that is widely found in citrus fruits [7]. Numerous studies have shown that limonin exhibits a wide spectrum of biological and pharmacological activities, including anti-cancer [8][9][10], anti-inflammatory [10,11], anti-oxidative [12], anti-viral [13], and liver-protective properties [14,15]. Hesperidin, or vitamin P, is a flavanone glycoside found in all types of citrus fruits. It is one of the safest and most valuable bioactive compounds, which possess a wide range of pharmacological properties [16]. Hesperidin has exhibited anti-type II diabetic [16], anti-inflammatory [17], anti-oxidative [17,18], anti-cancer [19,20], and cardioprotective properties [21,22], etc. These findings suggest that limonin and hesperidin can provide health-promoting benefits. Nevertheless, the traditional extraction methods for limonin and hesperidin usually require organic solvents that are toxic and harmful to the environment (e.g., dichloromethane, ethyl acetate, toluene, etc.) [23,24]. New extraction methods such as supercritical carbon dioxide (SC-CO 2 ) have been developed to solve this problem [25]. SC-CO 2 extraction reduces the organic solvent for extraction, resulting in a more environmentally friendly process. Nevertheless, this process requires high costs, sophisticated devices, and special expertise [24,25]. Thus, alternative extraction methods that use energy-saving plant sources and environmentally friendly bio-solvents are necessary. The present study is aimed at developing a green and simple extraction method to acquire the highest yield of both limonin and hesperidin from lime peel. Factors affecting extraction efficiency were studied, and the optimum green extraction method was proposed to maximize the use of food waste for appreciable cost-effectiveness and environmental sustainability.

Chromatograms and Mass Spectra of Limonin and Hesperidin
Chromatogram and mass spectra of limonin and hesperidin obtained from ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) are shown in Figure 1. The precursor mass of limonin was m/z 471 ( Figure 1A). The quantified and confirmed product ions of limonin were m/z 425 and 367, respectively ( Figure 1B). The precursor mass of hesperidin was m/z 611 ( Figure 1C). The quantified and confirmed product ions of limonin were m/z 303 and 465, respectively ( Figure 1D). The retention times of limonin and hesperidin were 0.6 and 1.6 min, respectively. Calibration curves showed good linearity with an average R 2 of 0.9932 and 0.9912 for limonin and hesperidin, respectively ( Figure 1E,F). Intra-day precision for limonin and hesperidin was 0.26-8.33% and 0.76-7.76% RSD, respectively. Inter-day precision for the analyses of limonin and hesperidin was 0.43-9.58 and 0.46-10.75, respectively.

Effect of Extraction Factors on Limonin and Hesperidin Concentration
As shown in Figure 2, the yields of limonin and hesperidin concentrations were varied upon ethanol concentrations and pH values. At pH 7 and 50 • C, extraction with 80% ethanol showed the highest yields among all ethanol concentrations tested. However, pH values of ethanol solutions also affected limonin and hesperidin concentration. The concentration of limonin and hesperidin tended to decrease when the pH value increased from 7 to 9 and from 7 to 11, respectively, for most ethanol concentrations, except for those of 100% ethanol at pH 9. Furthermore, temperature is also another factor affecting extraction efficiency. As shown in Figure 3, at pH 7 when the extraction temperature rises from 50 to 60 • C and from 50 to 70 • C, respectively, the yields of limonin and hesperidin Molecules 2022, 27, 820 3 of 12 concentrations seemed to decrease in most conditions, except for those of hesperidin after extraction with 100% ethanol (no changes upon temperature rise).

Effect of Extraction Factors on Limonin and Hesperidin Concentration
As shown in Figure 2, the yields of limonin and hesperidin concentrations were varied upon ethanol concentrations and pH values. At pH 7 and 50 °C, extraction with 80%  7 to 9 and from 7 to 11, respectively, for most ethanol concentrations, except for those of 100% ethanol at pH 9. Furthermore, temperature is also another factor affecting extraction efficiency. As shown in Figure 3, at pH 7 when the extraction temperature rises from 50 to 60 °C and from 50 to 70 °C, respectively, the yields of limonin and hesperidin concentrations seemed to decrease in most conditions, except for those of hesperidin after extraction with 100% ethanol (no changes upon temperature rise). (▼) ethanol in water with the specified pH values at 50 °C for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, < 0.01, < 0.001 and < 0.0001, respectively, compared with the concentrations of limonin or hesperidin at pH 7 of each specified condition. , and 100% (▼) ethanol in water with the specified temperatures, at pH 7 for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, < 0.01, < 0.001 and < 0.0001, respectively, compared with the concentrations of limonin or hesperidin at 50 °C of each specified condition.
As shown in Figures 2-3, regardless of pH or temperature, the use of mixed solvents (ethanol and DI water) provided higher yields of limonin and hesperidin than those of the pure solvent (100% ethanol). The highest concentrations of limonin and hesperidin were achieved by extraction with 70% and 80% ethanol (v/v), respectively. ( ) ethanol in water with the specified pH values at 50 • C for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, <0.01, <0.001 and <0.0001, respectively, compared with the concentrations of limonin or hesperidin at pH 7 of each specified condition. Figures 2 and 3, regardless of pH or temperature, the use of mixed solvents (ethanol and DI water) provided higher yields of limonin and hesperidin than those of the pure solvent (100% ethanol). The highest concentrations of limonin and hesperidin were achieved by extraction with 70% and 80% ethanol (v/v), respectively.

Optimization of Extraction Conditions
The results of this study showed that ethanol concentration, pH value of solvent, and extraction temperature were key factors affecting the yields of limonin and hesperidin. When analyzed with ANOVA, the interactions between these factors were found. Therefore, response surface methodology (RSM) was used to demonstrate the relationship between these factors and was used to identify the most influential factor. Figure 4A-C and Figure 4D-F show the RSM models for limonin and hesperidin concentration, respectively. The optimum values of the factors are shown within the warm tone color (maximum within red) regions. The response surface analysis (RSA) using ANOVA showed that the extraction yield of limonin depended most on ethanol concentration, followed by extraction temperature. The extraction yield of hesperidin depended most on extraction temperature followed by ethanol concentration. The pH value had the least effect on the extraction yields of both limonin and hesperidin.

Yields of Limonin and Hesperidin in Ethanolic-Aqueous Extracts of Lime Peel
The extraction yield was calculated as mg of limonin or hesperidin per gram of dry peel. As shown in Table 1, the condition yielding the highest amount of limonin was the extraction with 70% ethanol at pH 7 and 60 • C, while the condition yielding the highest amount of hesperidin was the extraction with 80% ethanol at pH 7 and 50 • C. For limonin, the extraction yields of 70% and 80% ethanol were not significantly different. However, for hesperidin, 80% ethanol provided a significantly better yield. Therefore, the optimal extraction condition achieving the highest amounts of limonin and hesperidin was 80% ethanol at pH 7, 50 • C. ethanolic-aqueous extracts of lime peel after extraction with 60% (•), 70% (■), 80% (▲), and 100% (▼) ethanol in water with the specified pH values at 50 °C for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, < 0.01, < 0.001 and < 0.0001, respectively, compared with the concentrations of limonin or hesperidin at pH 7 of each specified condition. , and 100% (▼) ethanol in water with the specified temperatures, at pH 7 for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, < 0.01, < 0.001 and < 0.0001, respectively, compared with the concentrations of limonin or hesperidin at 50 °C of each specified condition.
As shown in Figures 2-3, regardless of pH or temperature, the use of mixed solvents (ethanol and DI water) provided higher yields of limonin and hesperidin than those of the pure solvent (100% ethanol). The highest concentrations of limonin and hesperidin were achieved by extraction with 70% and 80% ethanol (v/v), respectively. ( ) ethanol in water with the specified temperatures, at pH 7 for 100 min. The statistical differences were separately analyzed for limonin and hesperidin by using one-way ANOVA followed by Tukey's multiple comparisons test. * , **, *** and **** mean p < 0.05, <0.01, <0.001 and <0.0001, respectively, compared with the concentrations of limonin or hesperidin at 50 • C of each specified condition.

Optimization of Extraction Conditions
The results of this study showed that ethanol concentration, pH value of solvent, and extraction temperature were key factors affecting the yields of limonin and hesperidin. When analyzed with ANOVA, the interactions between these factors were found. Therefore, response surface methodology (RSM) was used to demonstrate the relationship between these factors and was used to identify the most influential factor. Figures 4A-C and 4D-F show the RSM models for limonin and hesperidin concentration, respectively. The optimum values of the factors are shown within the warm tone color (maximum within red) regions. The response surface analysis (RSA) using ANOVA showed that the extraction yield of limonin depended most on ethanol concentration, followed by extraction temperature. The extraction yield of hesperidin depended most on extraction temperature followed by ethanol concentration. The pH value had the least effect on the extraction yields of both limonin and hesperidin.

Discussion
A number of scientific evidence supports the use of plant extracts to promote good health and wellbeing [26]. However, traditional methods for plant extraction are energyconsuming and environmentally unfriendly [1]. In this study, we demonstrated a good example of green extraction by utilizing food waste such as lime peel as a plant source and by using ethanol-water as a bio-solvent to achieve the highest amount of double bioactive compounds, i.e., limonin and hesperidin. LC-MS/MS was used to accurately determine the amount of bioactive compounds in the extract. The findings of this work suggest that ethanol concentration and extraction temperature were key factors affecting the extraction yields of limonin and hesperidin, while pH value had the least effect. The optimal extraction condition achieving the highest amounts of limonin and hesperidin was 80% ethanol at pH 7, 50 • C, which yields 2.072 and 3.353 mg/g of limonin and hesperidin, respectively.
Previous studies mostly used toxic organic solvents such as dimethyl sulfoxide (DMSO), dichloromethane, ethyl acetate, and toluene for plant extraction of limonin and hesperidin [23,24]. Instead, less toxic solvents, i.e., ethanol and water, were chosen for extraction in this study. In comparison with previous studies [12,27], the extraction yields of limonin and hesperidin in this work are relatively lower. The yield ranges for limonin and hesperidin in previous studies were 2.3-11.38 and 0.0-3.6 mg/g, respectively. The yield ranges for limonin and hesperidin in this study were 0.170-2.191 and 0.003-3.353 mg/g. Furthermore, the effect of pH on extraction yields, which was shown to be crucial in previous studies, plays a minor role according to our current study. One of the main reasons of the discrepancy is the difference in detection methods. Previous works mostly used high-performance liquid chromatography (HPLC) to detect limonin and hesperidin in plant products, including in citrus fruits [28]. However, the limitation of HPLC is its inability to separate compounds that have similar properties in interactions of the mobile and stationary phases [28][29][30]. Analysis of bioactive compounds using HPLC usually produced high yields, but they were not entirely specific. Therefore, HPLC mostly provides a rough estimate of the whole class of compounds (e.g., limonoids or flavanone) rather than the accurate determination of specific bioactive compounds (e.g., limonin and hesperidin). In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS) can determine the existence and the amount of specific compounds based on mass-to-charge ratio of ions and fragment ions that are produced during the ionization process [31]. In comparison with HPLC, LC-MS/MS provides a more accurate separation and quantification of specific bioactive compounds. Therefore, in this study, LC-MS/MS was chosen to specifically determine the amounts of limonin and hesperidin.
In general, the efficiency of plant extraction mainly depends on type of solvent, duration, pH value, and temperature [32]. Therefore, in this study, we investigated the impact of those factors. In the case of limonin, previous studies have reported that pH value had a significant effect on limonin concentration through the activity of Limonin-D-ring-lactonase (LDRLase) [24]. LDRLase is an enzyme found in citrus fruits that catalyzes the reversible conversion between limonin and limonoate A-ring lactone (LARL). The activity of LDRLase depends on pH value. In an alkaline condition (pH ≥ 8), LDRLase catalyzes a ring-opening reaction through hydrolysis of the D-ring in limonin and converts it into LARL [33]. LC-MS/MS is specific to limonin; therefore, the LARL form will not be detected at the same measurement. This fact may explain why the yield of limonin decreases when extracted at a higher pH. Nevertheless, according to our response surface methodology, pH value of extraction solvent had the smallest effect on limonin concentration, compared to ethanol concentration and extraction temperature. Likewise, pH value of extraction solvent had the least effect on hesperidin concentration. Previous studies have shown that the solubility of hesperidin significantly increased at higher pH. However, at a pH value greater than 9, degradation of hesperidin could also occur [34]. Thus, increasing the pH did not result in a significantly higher hesperidin concentration, when hesperidin was specifically determined by LC-MS/MS. In this study, the extraction temperature and ethanol concentration were shown to play an important role in the extraction efficiency of limonin and hesperidin from lime peel. For ethanol concentration, we found that the use of mixed solvents (ethanol and DI water) provided higher yields of limonin and hesperidin than those of pure ethanol. Consistently, a previous report by Gertenbach demonstrated that the presence of water in the solvent mixture causes the plant cells to swell, allowing easier diffusion of the solvent into the plant cells [35]. For extraction temperature, we found that an increase in the extraction temperature to higher than 50 • C actually reduced the yield of limonin and hesperidin. Generally, the solubility of bioactive compounds is increased with a rise in temperature; thus, heat is expected to increase extraction efficiency [36]. Nevertheless, the effect of temperature on extraction also depends on the chemical properties of the extracted compounds. In the case of limonin and hesperidin, previous studies reported that, as the temperature rises, limonin can significantly degrade [37], and hesperidin can be converted into its aglycone form (hesperetin) through chemical or enzymatic processes [34]. It was also reported that hesperidin glycosylated form and hesperidin complex has high thermal stability, but pure hesperidin has low thermal stability. These structural differences cannot be detected with the HPLC technique [38], but they can be distinguished with LC-MS/MS. In this study, we used LC-MS/MS to specifically determine only limonin and hesperidin. Therefore, temperature-dependent degradation or conversion can result in a decrease in limonin concentration when detected with LC-MS/MS. Future studies using LC-MS/MS are warranted to characterize various forms of limonin and hesperidin derivatives in varied extraction conditions. However, this study indicated that the pH values of extracts could be different from those of the solvents, which is consistent with a previous study by Hosseini that extracted from red cabbage, barberry, and eggplant peel. They reported that pH values of the extract may change after extraction and were found to differ when extracted with different solvents [39]. This may be the result of various extraction factors, such as types and percentage of the extraction solvent, properties of the sample, solid-to-solvent ratio, etc., that affect the extraction of acidic substances in the sample. Future investigation of this point is worthwhile.
Considering the interaction between several factors affecting extraction efficiency, our response surface methodology data is consistent with previous studies. Qin et al. showed that extraction temperature had a profound effect on the limonin concentration in the extract of pummelo seeds [12]. Another study, which extracted hesperidin from three types of citrus peels (orange, lemon, and clementine), found that ethanol concentration significantly affected hesperidin concentration in every sample, while the extraction temperature had a significant effect on some samples [40]. The most influential factors on extraction efficiency may vary depending on the species or part of the plant used for extraction. Furthermore, other factors, e.g., solid-to-solvent ratio, time for extraction, level of shaking, etc., also impact extraction yields. Therefore, to obtain consistently high yields of plant extract, optimization of the extraction method and good quality control of the extraction condition are absolutely required.
The strength of this work includes the detailed design of varied conditions for ethanol concentration, temperature, and pH to elucidate the interactions among these extraction factors. In addition, the use of LC-MS/MS allows for accurate and specific measurements of the bioactive compounds. Nevertheless, the limitations of this work include the fact that only temperatures of 50 • C and above and pH of 7 and above were tested. Furthermore, the lime peel powder used for extraction was randomly collected from the same batch of the industrial food waste. Future studies are warranted to compare the extraction yields of limonin and hesperidin from various batches of food waste. Calculation of the coefficient of variations (CV) is required to determine the consistency. Using industrial food waste, such as lime peel, from a standardized lime juice factory not only saves energy in growing new lime trees, but it also benefits from the good manufacturing practice (GMP) system to control the consistency of raw materials, i.e., the source of lime. Another limitation of this study is that we only used ethanol as an extraction solvent. The aim of this work was to develop a green extraction process. Therefore, ethanol was chosen based on its low cost, solubility, and environmentally friendly properties. This study used ethanol for extraction and LC-MS-MS for quantitation of extraction yields, while previous studies used more harmful solvents for extraction and HPLC for analysis. Therefore, the extraction yields obtained in this study cannot be fairly compared with previous works. Future studies are warranted to compare the extraction yields of ethanol with other types of solvents in parallel experiments and using similar analytical methods such as LC-MS/MS. This study discovered an optimized green extraction method to achieve the highest yields of limonin and hesperidin by using 80% ethanol in water to extract lime peel at pH 7 and 50 • C. The limonin-hesperidin-rich ethanolic-aqueous extracts of lime peel are expected to be highly effective owing to the dual bioactive compounds. Future in vitro and in vivo studies are warranted to investigate its health benefits such as anti-cancer, anti-inflammatory and anti-oxidative properties.

Experimental Design
Based on the chemical properties of limonin [41] and hesperidin [34], three variables, namely solvent dosage, pH of solvent, and temperature of extraction, were selected to vary the extraction conditions. Other factors, including time for extraction, the level of shaker and solid-to-solvent ratio, remained constant in all experiments. Ethanol was chosen as the extraction solvent in this study because it was a relatively low-cost organic solvent that classified as an environmentally friendly preferable green solvent and commonly used in various industries [42]. Lime peel powder was extracted with 60%, 70%, 80% ethanol in water, and 100% ethanol, and varied pH values of solvent at 7, 9, and 11. All extraction experiments were performed with a solid-to-solvent ratio of 0.01 g/mL in a shaking water bath (Memmert GmBH Co., Büchenbach, KG, Germany) at shaking speed level of 3.5 for 100 min. The extraction temperatures varied at 50, 60, or 70 • C. All conditions of extraction were performed three times separately, and three independent samples were used for data analysis. Response surface methodology was used to determine the optimum extraction conditions to extract limonin and hesperidin by varying operating parameters according to 3 × 3 factorials. Then, the concentrations of limonin and hesperidin were measured by UHPLC-MS/MS.

UHPLC-MS/MS Analysis
Measurement of limonin and hesperidin was performed with a method adapted from a previous study [43,44]. The extracts were collected and filtered through a 0.2 micron nylon syringe filter. The extracts were adjusted pH values to 7.4 with sodium hydroxide or citric acid solution. The final volumes were adjusted by using phosphate-buffered saline (pH 7.4) prior to analysis. The samples were analyzed with ultra-high performance liquid chromatography (UHPLC)-tandem mass spectrometry by using Ultimate 3000 connected with a TSQ Quantis Triple Quadrupole Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). UHPLC was performed by using Hypersil GOLD C18 column (1.9 µm particle size, 100 mm × 2.1 mm, ThermoScientific, Waltham, MA, USA). The column was maintained at 40 • C. Gradient run at a flow rate of 0.3 mL/ min for 5.10 min was performed using two types of mobile phase, i.e., 0.1% formic acid in deionized water (A) and 0.1% formic acid in methanol (B). The gradient program included: 0-1.3 min, 15-30% B; 1.30-2.50 min, 30-60% B; 2.50-5.10 min, 60-60% B. The injection volume was 1 µL. Tandem mass spectrometric analysis was performed in electron spray ionization with positive ion mode (ESI+) at spray voltage of 3.5 kV, ion transfer tube temperature at 350 • C, vaporizer temperature at 400 • C, sheath gas, auxiliary gas and sweep gas at 60, 15 and 2 arbitrary units, respectively. Mass spectrometer analyses of all samples were performed in selected reaction monitoring (SRM) mode for simultaneous analysis of multiple masses. The mass-to-charge ratio (m/z) of limonin precursor and quantified product mass were 471 and 425, respectively. The collision energy of 20 V was used for the transition. The confirmation product mass for limonin was m/z 367 with the collision energy of 19 V for the transition. For hesperidin, the precursor and quantified product mass were m/z 611 and 303, respectively. The collision energy of 20 V was used for the transition. The confirmation product mass for hesperidin was m/z 465 with the collision energy of 12 V for the transition.
Standard curves were generated from standard solution of single compound (limonin or hesperidin) at the concentrations of 5, 10, 25, 50, 100 µg/ mL in ethanol. The areas under the curve (AUC) of MS chromatograms for the quantified product mass and the corresponding concentrations of limonin and hesperidin were used to create linear standard curves.

Statistical Analysis
Statistical analyses were performed using PASW Statistics 18 (formerly SPSS Statistics) or GraphPad prism V.9. Data were expressed as means ± SD of at least 3 independent experiments (three separate extractions). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons or Ryan-Einot-Gabriel-Welsh F (REGWF) post hoc test was performed to compare the mean of each experimental condition. Multi-factorial ANOVA was used to determine the interaction effect of independent variables. A p value < 0.05 indicated a statistically significant difference.

Conclusions
This study demonstrated that lime peel obtained as food waste from a lime juice factory could be a potential renewable plant source for limonin and hesperidin-a rich extract. Using LC-MS/MS for determination of the bioactive compounds together with response surface methodology, we discovered that solvent concentration and extraction temperature were crucial factors that influenced extraction yields. The optimum condition to obtain the highest yields of limonin and hesperidin from lime peel was extraction with 80% ethanol at a solid-to-solvent ratio of 0.01 g/mL and pH 7 at 50 • C for 100 min. This condition provided the yields of limonin and hesperidin at 2.072 and 3.353 mg/g, respectively. The method used ethanol and water as the extraction solvent, which is safer for extractors and more environmentally friendly than other previously used organic solvents. Therefore, this process offers a good alternative that may be applied for implementation on an industrial scale as a green extraction process of limonin and hesperidin.