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Anim Biosci > Volume 34(4); 2021 > Article
Yang, Wang, Zhu, Li, and Ma: Degradation kinetics of vitamins in premixes for pig: effects of choline, high concentrations of copper and zinc, and storage time

Abstract

Objective

The present work was undertaken to evaluate the effects of storage time, choline chloride, and high concentrations of Cu and Zn on the kinetic behavior of vitamin degradation during storage in two vitamin premixes and four vitamin-trace mineral (VTM) premixes.

Methods

Two vitamin premixes (with or without 160,000 mg/kg of choline) were stored at 25°C and 60% humidity. Besides, four VTM premixes were used to evaluate the effects of choline (0 vs 40,000 mg/kg) and trace minerals (low CuSO4+ZnO vs high CuSO4+ZnO) on vitamin stability in VTM premixes stored in room, and the VTM premixes were stored in room temperature at 22°C. Subsamples from each vitamin and VTM premix were collected at 0, 1, 2, 3, 6, and 12 months. The retention of vitamin A (VA), vitamin D3 (VD3), vitamin E (VE), vitamin K3 (VK3), vitamin B1 (VB1), vitamin B2 (VB2), vitamin B3 (VB3), vitamin B5 (VB5), and vitamin B6 (VB6) in vitamin premixes and VTM premixes during storage was determined. The stability of vitamins in vitamin premixes and VTM premixes was determined and reported as the residual vitamin activity (% of initial) at each sampling point.

Results

The effect of choline on VK3 retention was significant in vitamin premixes (p<0.05). The negative effect of storage time was significant for the retentions of VD3, VK3, VB1, VB2, VB5, and VB6 in vitamin premix (p<0.05). For VTM premixes, negative effect of storage time was significant (p<0.05) for the losses of vitamin in VTM premixes. Choline and high concentrations of Cu and Zn significantly increased VA, VK3, VB1, and VB2 loss during storage (p<0.05). The supplementation of high concentrations of Cu and Zn significantly decreased the concentrations of VD3 and VB6 (p<0.05) in VTM premixes at extended storage time.

Conclusion

The maximum vitamin stability was detected in vitamin and VTM premixes containing no choline or excess Cu and Zn. The results indicated that extended storage time increased degradation of vitamin in vitamin or VTM premixes. These results may provide useful information for vitamin and VTM premixes to improve the knowledge of vitamin in terms of its stability.

INTRODUCTION

Vitamin and vitamin-trace mineral (VTM) premixes are designed for supplemental nutritional support to animals who are unable to ingest adequate amounts of natural feedstuffs [1,2]. Choline serves some essential biological functions in young animals [3,4], such as improving fat transport and metabolism in the liver, source of methyl donors for methionine regeneration from homocysteine, building and maintaining cell wall structure, and supporting nervous system function.
In the post-weaning period, the stress of being removed from the sow and mixing into a new environment results in perturbations of gut microbiota and lowered defenses against pathogen entry for piglets, which can lead to increased risk of disease [5,6]. Post-weaning diarrhea is one of the most common causes of mortality for weanling pigs, and hence greatly impaired growth performance of pigs [5,6]. In practical application, the inclusion of high concentrations of zinc oxide (ZnO) and copper sulfate (CuSO4) in weaned pig diets can improve the growth performance and decrease diarrhea incidence [57]. All of these treatments may affect the formulation of the ingredients in premixes, leading to bioavailability loss of necessary nutrients. In addition, premixes are often not consumed immediately after manufacturing and can be degraded due to several factors, such as the length of storage and premix compositions. (e.g. choline chloride and high concentrations of metal ions) [3,811]. Numerous reports have discussed the stability of vitamins under various conditions such as storage and processing [811]. Vitamins may be unstable to heat and light as well as exposure to acid, alkali, air, and moisture. The degradation of vitamin activity in premixes and complete feed during storage may result in hidden depressions in growth, feed efficiency, and disease resistance due to the subclinical vitamin deficiencies [3,8,11]. For this reason, the vitamin content should be evaluated after manufacturing and storage in order to ensure the amount provided.
However, studies on the stability of vitamins in vitamin premix and VTM premix are limited. In addition, these factors (choline chloride and high concentrations of CuSO4 and ZnO) in premixes have received limited research attention, and it is unclear which vitamins in vitamin or VTM premixes are vulnerable when choline and high concentrations of Cu and Zn are present. Therefore, the objectives of this study were to i) determine the rate of vitamin loss in vitamin or VTM premixes and the effects of choline and high concentrations of Cu and Zn on the stability of vitamins during storage and ii) to develop vitamin retention prediction models based on storage time.

MATERIALS AND METHODS

Experimental design

This study was conducted at the State Key Laboratory of Animal Nutrition at China Agricultural University (Beijing, China) and Ministry of Agriculture and Rural Affairs Feed Efficacy and Safety Evaluation Center (Beijing, China). Approval from the Animal Care and Use Committee was not obtained for this experiment because no animals were used.

Chemical reagents

Deionized water (18 Mω·cm) from a Millipore Milli-Q (Bedford, MA, USA) water purification system was used to prepare all aqueous solutions. Standard for retinyl esters, cholecalciferol, α-tocopherol acetate, menadione, thiamine, riboflavin, pyridoxine, niacin and pantothenic acid were purchased from Sigma–Aldrich (Fluka, Sigma–Aldrich, Steinheim, Germany). The methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade were used for HPLC analysis and were obtained from Fisher Scientific (Pittsburg, PA, USA). All the other chemicals used were analytical grade and purchased from Sinopharm Chemical Reagent LTD (Beijing, China).

Premix formulation and treatments

Two vitamin premixes (containing no trace minerals) were formulated by commercial vitamins. The vitamin manufacturer is not disclosed in order to protect proprietary information. The manufacturing dates of all vitamins were obtained from the original suppliers to ensure that the products were within 6 months of manufacture and were not expired. The vitamin premixes were designed to be added at a rate of 2.5 g/kg of the diet. The specific vitamin composition and inclusion level were set to mimic common swine industry vitamin premixes. One of vitamin premixes contained choline, another vitamin premix without choline (Table 1). The choline chloride concentration in vitamin premix was measured via chromatography [12].
Four VTM premixes were formulated to contain the same level of vitamins for weanling piglets. The VTM premixes were designed to be added at a rate of 10 g/kg of the diet, which is common in practice. Compositions of VTM premixes were shown in Table 1. Vitamin levels met or exceeded the requirement of NRC [13], which were chosen to represent the “typical” industry levels based on informal surveys of vitamin levels in commercially available premixes. The amounts of each vitamin used in each premix are shown in Table 2. Choline was added to VTM premixes 2 and 4. The choline chloride concentration in VTM premix was determined using chromatography [12]. The VTM premixes 1 to 4 were formulated to meet or exceeded NRC requirements for copper (Cu), iodine (I), iron (Fe), manganese (Mg), selenium (Se), and zinc (Zn) for piglets [13]. The VTM premixes 3 and 4 contained 20,000 mg/kg of Cu added as CuSO4 and 225,000 mg/kg per diet of Zn added as ZnO, which could provide 200 mg/kg of Cu and 2,250 mg/kg Zn in diet. The reason for choosing these levels of Cu and Zn is that weanling pig premixes commonly have higher concentrations of Cu and Zn as an antimicrobial and improve growth performance [57].

Premix preparation and storage

Vitamin and VTM mineral premixes were manufactured at a commercial vitamin premix plant. Each of the two vitamin premixes was prepared in six separate batches using an identical procedure (7.2 kg per batch). Each batch represented one replicate and was divided into six-1.2 kg, thick polyethylene bags with plastic ties. Each of the four VTM premixes was prepared in six separate and identical procedure (18 kg per batch). Each batch represented one replicate and was divided into six-3.0 kg, thick polyethylene bags with plastic ties. Vitamin premixes were stored in a controlled environment chamber setting at 25°C and 60% relative humidity. The VTM premixes were stored in a storage room (22°C).

Vitamin sampling, extraction and assays

Subsample of vitamin and VTM premixes were obtained from each of six replicates at 0, 1, 2, 3, 6, and 12 months. Samples were immediately sent to the Ministry of Agriculture and Rural Affairs Feed Efficacy and Safety Evaluation Center (Beijing, China) used for vitamin analysis. The vitamin A (VA) and E (VE) were determined by the method 2012.10 [14]. In brief, the sample (2 g) was mixed with papain solution until dispersed, placed in a 37°C±2°C water bath, and extracted by methanol. This extract was analyzed by HPLC (Agilent 1200 Series; Agilent Technologies Inc., Santa Clara, CA, USA). For the extraction of vitamin D3 (VD3) from samples, the method of 992.26 [14] was used. In brief, 5 g sample was transferred to a centrifuge tube and anhydrous ethanol, ascorbic acid, and potassium hydroxide were added. Tubes were placed in a 75°C water bath. Subsamples were analyzed by HPLC followed by UV detection at 254 nm. For the determination of vitamin K3 (VK3), samples were extracted with trichloromethane. Extract filtered and injected into the HPLC system and UV detection was made at wavelength 251 nm [15]. For the extraction of water-soluble vitamins (vitamin B1 [VB1], vitamin B2 [VB2], vitamin B3 [VB3], vitamin B5 [VB5], and vitamin B6 [VB6]) from diets, the procedure of Chen et al [16] was used and modified. Five g of sample was weighed, extracted with phosphate buffer, heated in a water bath, and sonicated. The supernatants of the extracted samples were stored at −20°C until they were tested. These extracted samples were analyzed using a 250× 4.5 mm, 5 μm, Eclipse Plus C18 column (Agilent Technologies Inc., USA) on an Agilent liquid chromatograph. The stability of vitamins in vitamin premixes and VTM premixes during storage was determined and reported as the residual vitamin activity (% of initial) at each sampling point. This time was also convenient for us to compare with previous studies and for developing predicted equations to estimate the vitamin loss. The specific vitamin selected were based on the capacity to complete our respective lab analysis.

Data treatment and statistical analysis

Normality of the data was verified using the UNIVARIATE procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The BOXPLOT procedure of SAS was used to check for outliers. Data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., USA) to determine the interactive and main effects of choline chloride, high concentrations of Zn and Cn, and storage time on the activity of vitamins in vitamin and VTM premixes. Results were considered significant at p≤0.05 and a tendency at p≤0.10. Diagrams were generated using the Excel 2016 (Microsoft Corporation, Redmond, WA, USA). Vitamin retention was modelled using linear regression and non-linear regression. Linear regression to determine vitamin retentions was completed using the PROC REG procedure of SAS. Non-linear regression was performed using PROC NLIN procedures of SAS with exponential model (Eq. (1)). The exponential model is flexible owing to the inclusion of a shape constant in addition to the rate constant and has been employed to describe vitamin degradation kinetics [17].
(1)
Ct=C0×e-Kaat
Where Ct is the vitamin concentration at a time t, C0 is the initial vitamin concentration, Kaa is the rate constant. Modelling and analysis of variance were performed using SAS 9.4 (SAS Inst. Inc., USA). The R2 and the root mean square error of prediction (RMSEP) were used to define the best-fit equations.

RESULTS

Vitamin recovery method validation

Methods for vitamin analysis in samples were validated with repeatability between-day precision, long-term precision, limits of quantitation, and linearity (data not shown) by the staff of the Ministry of Agriculture and Rural Affairs Feed Efficacy and Safety Evaluation Center (Beijing, China). Calculated values (Table 1) were determined from the minimum declared vitamin concentrations as provided by each product’s manufacturer. The initial (d 0) analyzed vitamin concentrations of the vitamin premix, or VTM premixes are reported in Table 2. The analyzed vitamin values in vitamin premix or VTM premixes were similar to calculated vitamin values in vitamin premix or VTM premixes.

Effects of storage time and choline on stability of vitamins in vitamin premixes

There was no significant interactive effect of storage time and choline on VA, VD3, VE, VB1, VB2, VB3, VB5, and VB6 retention (Table 3). The main effect of storage time was significant (p<0.01) for the retentions of VD3, VB1, VB2, VB5, and VB6 in vitamin premix (Figures 1 to Figure 2Figure 3Figure 45). The retention of VK3 was significantly influenced by storage time (p<0.01), choline (p<0.01) and the interaction between storage time and choline (p<0.01; Table 3; Figure 6).
At 3 months, VA, VD3, VE, VK3, VB1, VB2, VB3, VB5, and VB6 retained at least 96%, 92%, 97%, 78%, 95%, 94%, 98%, 94%, and 90% of their initial activity in vitamin premix without choline, respectively. At 12 months, most vitamins retained 76% to 98% of the initial vitamin activity, but VK3 was retained 47.64% of its initial activity in vitamin premix without choline. With increased storage time, VA exhibited a tendency (p = 0.096) for decreased retention in vitamin premixes, but VA exhibited a tendency (p = 0.076) and VB5 was marginally significant (p = 0.053) for decreased retention during extended storage time in vitamin premixes containing choline chloride.

Effects of storage time, choline, and Cu and Zn elements on stability of vitamins in VTM premixes

There were interactive effects among storage time, choline chloride and high concentrations of Cu and Zn on VK3, VB1, and VB2 retentions (p<0.05) (Table 4). Moreover, there were interactive effects between storage time and choline chloride on VA, VK3, VB1, and VB2 retentions (p<0.05). There were interactive effects between storage time and high concentrations of Cu and Zn on VA, VD3, VK3, VB1, VB2, and VB6 retentions (p<0.01). As well, there were interactive effects between choline chloride and high concentrations of Cu and Zn on VK3 retention (p<0.01). The main effect of storage time on all tested vitamins was significant (p<0.01), the main effect of choline was significant for VA, VK3, VB1, and VB2 retentions (p<0.01) (Figures 2, 3, 6, and 7), and the main effect of high concentrations of Cu and Zn on VA, VD3, VK3, VB1, VB2, VB6 retentions was significant (p<0.01) (Figures 1, 2, 3, 5, 6, and 7).
Extended storage time had negative (p<0.01) effects on vitamin stability in VTM premix. When VTM premixes were stored, VE, VB3, and VB5 activities decreased (p<0.05) (Figures 4, 8, and 9) as the duration of storage increased regardless of the choline chloride or high concentrations of Cu and Zn. Also, stability of VA, VK3, VB1, and VB2 was decreased in VTM premix containing choline and high concentrations of Cu and Zn. However, the activity of VB6 was rapidly decreased in VTM premix containing high concentrations of Cu and Zn (Figure 5).

Kinetic properties of vitamin retention

The R2, RMSEP, and the prediction equations are presented (Table 5). Analysis of kinetic data suggested that the degradation follows a first-order model. Degradation of VA, VD3, VK3, VB1, and VB6 in VTM premix 1 could be suitably modeled based on high R2 value (i.e. R2>0.90) of prediction equations. For premix containing chloride choline, equations for predicting retention of VA, VD3, VE, VK3, VB3, and VB6 in VTM premix 2 were suitably modeled based on high R2 value (i.e. R2>0.90), and those had high R2 values compared to equations predicting VB1, VB2, and VB5. While the ability to predict degradation of VB2 with storage was low (R2 = 0.492). In VTM premix 3, equations for predicting retention of VA, VD3, VK3, and VB5 had high R2 values (i.e. R2>0.90) and thus good fit to predict change in retention with storage time. However, similar ability to predict VB2 degradation with storage time was low (R2 = 0.477). In VTM premix 4, the equations had high R2 (i.e. R2>0.90) for predicting retention of VD3, VK3, VB1, and VB5 compared to predicting other vitamins.

DISCUSSION

Extended storage time affected the stability of vitamins

Determination of vitamin stability before and after storing is necessary to assess the final amount of actual vitamin that will reach the end-user and to calculate what amount should be added to any feed matrix. Storing vitamins for long periods has been considered to negatively affect vitamin activity in vitamin or VTM premixes. However, the results of our study indicate that long-term storage of vitamin premixes had little influence upon VA, VE, and VB3 concentrations. The results from our study agreed with previous studies on vitamin stability. Coelho [8] reported that the average loss per month of VA, VE, and VB3 after storage was 2.9%, 2.7%, and 2.7%, respectively. Because most manufactures of VA producers stabilize VA using the method of sealing within a physical matrix, generally arabic gum or gelatin [2,18]. In addition, in the production of commercially-available VA and VE, their hydroxy group is protected by the formation of an ester, as in α-tocopherol acetate. The obtained α-tocopherol acetate is resistant to oxygen, since it lacks double bonds [3,9]. In the present study, the retention of niacin was more than 90% after 1 year of storage, which is consistent with Zhuge and Klopfenstein [19], who reported that the retention rate of VB3 during storage was 91% to 96% during 27 weeks storage. This was not surprising to us because VB3 has been reported to be the most stable among the B vitamins when added to feed or premixes [19] owing to its a stable molecular structure, which reduces its oxidation during storage. Coelho [8] reported that the loss of commercial fat-soluble vitamins after one month of storage was less than 10%; after six months of storage, its loss rate was 10% to 60%. Shurson et al [20] reported that vitamin activity in vitamin and VTM premixes decreased with prolonged storage time, but stability of VE, VB2, VB3, VB5, and VB6 are higher than that of other vitamins, which is in line with our results.

Addition of choline chloride affected vitamin stability

Choline chloride has been reported to significantly affect vitamin activity [810]. Choline is considered a stress agent that affects vitamins that dissolve easily in water because it is hygroscopic, and can attract moisture to vitamin or VTM premixes [3]. The concentration of choline chloride in feed is usually higher than the micro-ingredient level, and problems related to both physical and chemical properties can be expected when choline is added to vitamin or VTM premixes [810]. After six months of storage, the loss of vitamin in a vitamin premix without choline chloride was 1% to 5% [8]; with choline chloride in the premix, the vitamin loss up to 32% after six months of storage [8]. In addition, the negative effects of choline chloride on vitamins in a VTM premix were more significant. After six months of storage, the loss of vitamin in a VTM premix without choline chloride was 12% to 30% [8]. However, in a VTM premix with choline chloride, the loss after six months was 23% to 52% [8]. Vitamin K is known for its contribution to the blood clotting or coagulation process. The VK3 was more stable in vitamin premix without choline than in the vitamin premix with choline during one-year storage, which was consistent with Tavčar-Kalcher and Vengušt [10]. Results from the present study suggest that loss of VK3 activity was approximately 60% in two vitamin premixes. Our results are similar to the average monthly losses reported by Coelho [8]. In the current study, choline chloride is a significant factor affecting the loss of VK3 activity. Menadione (VK3) is the form of vitamin K that is used in animal nutrition. It is not utilized in pure form in premix plants but was formulated with sodium bisulfite and derivatives. The most common menadione compound used in the industry is the water-soluble salt, menadione sodium bisulfite. It was reported that VK3 was very sensitive to moisture and trace minerals, and choline chloride was particularly destructive to VK3 [10]. Furthermore, supplemented choline chloride in vitamin or VTM premixes increased leaching of VK3 and prolonged oxidation-reduction reactions [9,10]. The inclusion of choline chloride in a VTM premix contributes to VA, VK3, VB1, and VB2 instability during storage. Stability data published by BASF 1994, cited by Whitehead [21], showed VA, VK3, VB1, and VB2 loss of 15%, 36%, 30%, and 5% after one month and 42%, 100%, 73%, and 44% after six months of storage in VTM premixes containing choline chloride. Besides, we observed that loss of VA, VK3, VB1, and VB2 was lower than that reported by Whitehead [21]. The reason may be that the vitamin manufacturing industry has developed products with improved stability. In the present study, we used thiamine mononitrate as VB1 source, it is used more often in feed because of its higher stability compared to thiamine hydrochloride [3,8]. In addition, the commercial form of VA and VB2 is spray-dried processing, which usually provides improved stability during storage and transportation.

Excessive Cu and Zn elements in VTM premix affected vitamin stability

The VTM premixes are the most common dietary supplements. In commercial conditions, feeding piglets with high concentrations of Zn and Cu stimulates their average daily gain, decreases the feed conversion ratio, improves the digestibility of dietary nutrients and growth performance, and decreases the incidence of diarrhea [6,7]. However, minerals in premixes are usually in an inorganic form: sulfates, chlorides, oxides, etc. Different metal compounds have different capability of catalytic oxidation reaction [8]. In the present study, high levels of CuSO4 (more than 20,000 mg/kg of Cu in premix to promote growth) and ZnO (more than 225,000 mg/kg of Zn in premix to decrease the incidence of diarrhea) in VTM premixes resulted in the degradation of vitamins. Vitamin stability is reduced in the presence of certain trace minerals [8,9,20]. In our study, blending vitamins with trace minerals to form VTM premixes increased the loss of vitamin activity during prolonged storage periods. These trace minerals in premix can catalyze the generation of free radicals, thereby oxidizing antioxidants during storage. Certain metal-catalyzed destruction of vitamins in the feed matrix has been reviewed previously to a limited extent [22]. Trace minerals vary in their redox potential: Cu, Fe, and Zn are the most reactive, and Se, I, and Mg are less reactive minerals [8,22]. Reactive trace minerals reduce vitamin activity by oxidizing the vitamins. First, the metallic-like nature of trace minerals reduces the crystals of vitamins to smaller particles by eroding their protective coating. The smaller particles provide increased surface areas of vitamins for reactions between vitamin particles and trace mineral particles.
Dove and Ewan [23] reported that a high concentration of Cu (250 mg/kg feed) or Zn (1,000 mg/kg feed) increased vitamin loss. Redox-active transition metals, such as Cu, can serve as catalysts for the oxidation of organic compounds. Lu et al [24] reported that a high concentration of Cu sulfate promoted the undesirable oxidation of VE in feeds. Intriguingly, we did not find significant effects on VE content in the four VTM premixes during long-term storage. There are two significant factors that might contribute to achieve this positive characteristic. First, supplementation of VE are generally given in the form of all-rac-α-tocopheryl acetate in which the reactive hydroxyl group of α-tocopherol is esterified; rendering the molecule more stable than the free phenol form, and second, production of VE provides a physical barrier to reduce surface contact with pro-oxidant agents such as Zn or Cu. On the other hand, we observed long-term storage of VTM premix with high concentrations of Cu and Zn had no effect on VB3 and VB5 concentrations. The results from our study confirm previous studies on the stability of these vitamins. Shurson et al [20] reported recovery of VB3 after 120 days of storage in a vitamin stock was 96.68%, 86.04% in vitamin premix, and 87.04% in VTM premix. Zhuge and Klopfenstein [19] also reported that VB3 was considerably more stable than other vitamins; at the end of 27 weeks storage, VB3 in premixes with or without mineral retained 91% and 96%, respectively. The VB3 is probably the most stable of the water-soluble vitamins when added to feed or premixes, being little affected by heat, oxygen, moisture, or light [3,11]. However, there are no reports on the mechanism of resistance to degradation or subsequent degradation products. The VB5, pantothenic acid, is a constituent of coenzyme A, which both act as carriers of acyl groups and activators of carbonyl groups in many metabolic processes [2,3]. Pure VB5 is a viscous, hygroscopic, and chemically-unstable oil. In premixes, this vitamin is commonly added as calcium pantothenate, a soluble and stable solid. The calcium salt is preferred to the sodium salt, as solid forms of the sodium salt are much more hygroscopic. Similar to niacin, there were no significant influences of choline and high concentrations of Cu and Zn on pantothenic acid retention in VTM premixes. But storage time was a significant factor affecting the loss of VB5 under ambient temperature and relative humidity [3]. According to the previous results [20], there was no significant difference in the stability of VB5 after 120 days of storage at vitamin stock, vitamin premix and vitamin-inorganic trace minerals premix. A similar result was reported by Coelho [8]. Furthermore, it was reported that pantothenic acid was relatively stable to heat, oxygen, and light [8]. The stability of pantothenic acid is due to the presence of the carboxylic acid group to form two hydrogen bonds between a pair of molecules [25]. Furthermore, the amide group and two methyl groups in the aliphatic chain of pantothenic acid contribute to its stability.
In the current study, the supplementation of high concentrations of Cu and Zn significantly reduced the concentrations of VA, VD3, VK3, VB1, VB2, and VB6 in VTM premixes during storage. The reason can be explained that the presence of Zn and Cu in premix can speed up vitamin degradation [3,8, 9], these minerals can catalyze the generation of free radicals which can oxidize vitamins in VTM premixes. Also, the results from our study confirm previous studies on vitamin stability in VTM premix. Shurson et al [20] reported that the stability of VA, VK3, VB1, and VB6 in premixes was influenced by the presence of trace minerals. These results were in line with previous study. Yang et al [9] reported that stability of VA, VB1, and VB6 was affected by trace minerals, and the premixes containing high levels of Cu and Zn was more susceptible to oxidative reaction of vitamins. Factors that reduce VA activity are atmospheric oxygen, light, heat and other oxidizing agents [3]. Manan et al [26] looked at the stability of VA in the presence of Cu and Zn; the results showed that mineral fortification reduced VA stability by 36%. Pinkaew et al [27] reported the stability of VA in the presence of ZnO; the results showed that mineral fortification reduced the VA stability by 13.4%. The chemical structure of VA is an unsaturated monohydric alcohol with 20 carbon atoms, consisting of a cyclohexane ring linked to a polyunsaturated chain that terminates in an alcohol group. The five conjugated double bonds in the configuration of VA are easy points of attack for oxygen. The oxidation of the alcohol end group of VA results in the formation of retinal or all-trans retinaldehyde, which can be further oxidized to all-trans retinoic acid. Besides, the structure of VB1 can help to understand its instability during storage. The methylene bridge connecting the pyrimidine and thiazole moiety can easily be broken down by oxidizing ingredients [25]. In addition, the VB6 comprises a group of three related compounds: pyridoxine, pyridoxal, and pyridoxamine. Pyridoxine is commonly used for feed because pyridoxine is more stable than either pyridoxal or pyridoxamine [3,8], but pyridoxine was sensitive to light, particularly in neutral and alkaline solutions. The VB6 can lose bioactivity, particularly when minerals in the form of carbonates or oxides are present [8,11]. In the current study, the supplementation of high concentrations of Cu and Zn significantly reduced the concentrations of VB6 in VTM premixes during storage. The loss of VB6 activity after three months of storage at room temperature was 24% [11], which was slightly higher than VB6 loss in the present study. It may be that the degradation reaction of VB6 was enhanced by metal ions in the previous study. Loss of VB6 was lower when stored as vitamin premixes compared to VTM premixes, maybe the reason was that VTM premix contained more trace mineral. In VTM premixes, VB6 can lose bioactivity, particularly when minerals in the form of carbonates or oxides are present [8,11]. We used pyridoxine hydrochloride in the present trial, which is a commercially available form. And pyridoxine hydrochloride is the main supplement used in feed, because it has good handling properties and stability. In addition, Coelho [8] reported the loss of VB6 after six months of storage in a vitamin premix was 17%, and 32% in a VTM premix. Although the retention rate of VB6 in Coelho’s study [8] was completely inconsistent with our results, our data also show that the stability of VB6 is reduced in the premix containing inorganic trace minerals.
There are very limited stability data available for VD3 and VB2 in VTM premixes. The supplementation of high concentrations of Cu and Zn decreased the stability of VD3 and VB2 in VTM premixes in the present study. The VD3 is a fundamentally unstable compound containing double bounds that can be altered by different stresses and prone to degradation due to oxidation [3,28]. The stability of VD to oxidation and its instability to trace minerals were also reported by previous studies; Mahmoodani et al [28] demonstrated catalyzed isomerization of VD3 is liable via autoxidation to form a variety of oxidation products. Further, Zhuge and Klopfenstein [19] reported that VB2 was destroyed faster in the premix containing minerals and 54% of VB2 had been destroyed after 27 weeks of storage. In the last case, the loss of VB2 after six months of storage was 44% cited by Whitehead [21]. The most well-characterized aspect of riboflavin reactivity is its sensitivity to light in an aerobic environment which may be one of the reasons for low VB2 retention of previous studies. The rate of degradation can be promoted by complexation with some metal cations (e.g., Cu2+ and Zn2+) at the isoalloxazine moiety [29]. Vitamin A, VD3, VK3, VB1, VB2, and VB6 are relatively stable in vitamin premix, but degradation of vitamin was potentiated by the chemical reaction caused by the presence of Cu and Zn elements.

Prediction equations for vitamin retention in premixes during storage

Prediction equations have been widely used to estimate values through regression analysis and can be a suitable proxy for conducting relevant experiments (i.e. loss in vitamin stability with time) and thus save time, reduce cost, and improve precision in diet formulation. To establish prediction equations of vitamin content in premix from storage time, regression analysis programming was used. The degradation of most of the vitamins followed first-order kinetics that can be developed [9,30]. The degradation rate accelerated progressively with storage time in vitamin and VTM premixes. What is more, VA, VK3, VB1, VB2, and VB6 degraded rapidly in VTM premix containing choline and high concentrations of Cu and Zn. When comparing the relative retention of vitamins, the data showed a significant deviation in different VTM premix. The VTM premix 4 has the most prominent sensitivity, followed by VTM premix 3 and 2. These observations are in line with previous report [9], showing poor vitamin retention in VTM premix containing choline and trace minerals. This differing behavior regarding vitamin degradation can be attributed to difference premix formulation and the presence of more metal ions, such as Cu2+ and Zn2+ that act as catalysts. The degradation of vitamins does not always increase linearly during storage, and the majority of predicted equations had good fit with the experimental data. This study is consistent with Giannakourou et al [30] who also found vitamin loss due to storage time was well predicted by exponential models. For prediction of vitamin loss during storage with non-linear kinetic models, this method has been proved effective to extract the kinetics parameters for vitamin degradation [31]. To the best of our knowledge, the degradation kinetics of vitamin in VTM premixes had not been studied before. The purpose of present study was to establish kinetic equations for vitamin retention in different premixes during storage. Validated kinetic models of vitamin for VTM premix, can be used for evaluation, control and proper management of the premix, with the application of suitable time indicators. Further, these results can serve for vitamin and VTM premixes production in order to improve the knowledge of vitamin its stability.

CONCLUSION

The present study has provided information on the kinetics of vitamin degradation in different premixes during storage. The degradation of vitamins in all the samples under all storage conditions followed the first-order kinetics. Stability of VA, VK3, VB1, VB2, and VB6 was higher in VTM premixes containing no choline or high concentrations of Cu and Zn. The high concentration of trace minerals in the VTM premix can negatively affect VD3 stability. Our study also suggested that considering the potential losses of vitamins in formulation, the time between the manufacturing and use of the vitamin premixes should be minimized, and vitamins and trace mineral premixes should be stored separately. This work could be used as a guideline for the fortification of vitamin and VTM premixes.

Notes

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Zhu M is an employee of Liyuan Group.

ACKNOWLEDGMENTS

This study was financially supported by the China Agricultural University Research Funding (201705510410056) and Ministry of Agriculture and Rural Affairs Funding (21178259). The authors express their appreciation to the staff of Ministry of Agriculture and Rural Affairs Feed Efficacy and Safety Evaluation Center (Beijing, China) for sample analyses. We want to thank Prof. Crystal Levesque in South Dakota State University for revising the manuscript. Also, I want to show my appreciation for Eason Chan; when a grey person walking a grey wall in a grey campus, the melody ‘Ren Lai Ren Wang, Tao Tai, and Bitter Gourd’ grew and passed my soul; Thanks to rekindle my heart in the dark.

Figure 1
Residual vitamin D3 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, p<0.001; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, NS; time× choline, NS; time×Cu/Zn, p<0.001; choline×Cu/Zn, NS; time, p<0.001; choline, NS; Cu/Zn, p<0.001).
ajas-20-0026f1.jpg
Figure 2
Residual vitamin B1 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline: NS; time, p<0.001; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, p = 0.013; time×choline, p<0.001; time×Cu/Zn, p<0.001; choline×Cu/Zn, NS; time, p<0.001; choline, p<0.001; Cu/Zn, p<0.001).
ajas-20-0026f2.jpg
Figure 3
Residual vitamin B2 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, p<0.001; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, p = 0.009; time×choline, p<0.001; time×Cu/Zn, p = 0.001; choline×Cu/Zn, NS; time, p<0.001; choline, p<0.001; Cu/Zn, p<0.001).
ajas-20-0026f3.jpg
Figure 4
Residual vitamin B5 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, p<0.001; choline, p = 0.053). The p-values in VTM premix (time×choline×Cu/Zn, NS; time×choline, NS; time×Cu/Zn, NS; choline×Cu/Zn, NS; time, p<0.001; choline, NS; Cu/Zn, NS).
ajas-20-0026f4.jpg
Figure 5
Residual vitamin B6 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, p<0.001; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, NS; time× choline, p = 0.052; time×Cu/Zn, p<0.001; choline×Cu/Zn, NS; time, p<0.001; choline, NS; Cu/Zn, p<0.001).
ajas-20-0026f5.jpg
Figure 6
Residual vitamin K3 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, p<0.001; time, p<0.001; choline, p<0.001). The p-values in VTM premix (time×choline×Cu/Zn, p = 0.002; time×choline, p<0.001; time×Cu/Zn, p<0.001; choline×Cu/Zn, p<0.001; time, p<0.001; choline, p<0.001; Cu/Zn, p<0.001).
ajas-20-0026f6.jpg
Figure 7
Residual vitamin A activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, NS; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, NS; time×choline, p = 0.011; time×Cu/Zn, p<0.001; choline×Cu/Zn, NS; time, p<0.001; choline, p<0.001; Cu/Zn, p<0.001).
ajas-20-0026f7.jpg
Figure 8
Residual vitamin E activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, NS; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, NS; time×choline, NS; time×Cu/Zn, NS; choline×Cu/Zn, NS; time, p<0.001; choline, NS; Cu/Zn, NS).
ajas-20-0026f8.jpg
Figure 9
Residual vitamin B3 activity (% of initial) for vitamin premixes and vitamin-trace mineral (VTM) premixes as affected by choline chloride, high concentrations of copper (Cu) and zinc (Zn) and time (1 to 12 months). Each data point is the mean of 6 observations. The p-values in vitamin premix (time×choline, NS; time, NS; choline, NS). The p-values in VTM premix (time×choline×Cu/Zn, NS; time×choline, NS; time×Cu/Zn, NS; choline×Cu/Zn, NS; time, p<0.001; choline, NS; Cu/Zn, NS).
ajas-20-0026f9.jpg
Table 1
Composition of the vitamin and vitamin-trace mineral premixes1)
Item Vitamin premix 1 Vitamin premix 2 VTM premix 1 VTM premix 2 VTM premix 3 VTM premix 4
Vitamin2) (unit/kg)
 VA (IU) 5,400,000 5,400,000 1,350,000 1,350,000 1,350,000 1,350,000
 VD3 (IU) 1,200,000 1,200,000 300,000 300,000 300,000 300,000
 VE (IU) 12,000 12,000 3,000 3,000 3,000 3,000
 VK3 (mg) 1,200 1,200 300 300 300 300
 VB1 (mg) 1,200 1,200 300 300 300 300
 VB2 (mg) 2,400 2,400 600 600 600 600
 VB3 (mg) 12,000 12,000 3,000 3,000 3,000 3,000
 VB5 (mg) 7,200 7,200 1,800 1,800 1,800 1,800
 VB6 (mg) 1,200 1,200 300 300 300 300
 Folic acid (mg) 48 48 12 12 12 12
 Biotin (mg) 12 12 3 3 3 3
 Vitamin B12 (mg) 9.6 9.6 2.4 2.4 2.4 2.4
 Choline (mg) - 160,000 - 40,000 - 40,000
Trace mineral3) (mg/kg)
 Cu - - 500 500 20,000 20,000
 I - - 14 14 14 14
 Fe - - 10,000 10,000 10,000 10,000
 Mg - - 300 300 300 300
 Se - - 25 25 25 25
 Zn - - 8,000 8,000 225,000 225,000

VTM, vitamin-trace mineral; VA, vitamin A; VD3, vitamin D3; VE, vitamin E; VK3, vitamin K3; VB1, vitamin B1; VB2, vitamin B2; VB3; vitamin B3; VB5, vitamin B5; VB6, vitamin B6.

1) Vitamin premixes were designed to be added at a rate of 2.5 g/kg of the diet. The VTM premixes were designed to be added at a rate of 10 g/kg of the diet. Vitamin premix 1 contained no choline chloride, Vitamin premix 2 contained 160,000 mg/kg of choline chloride. VTM premix 1 contained 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO, VTM premix 2 contained 40,000 mg/kg of choline chloride, 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO. VTM premix 3 contained 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO. VTM premix 4 contained 40,000 mg/kg of choline chloride, 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO. Limestone and rice by-product as carriers mix purified forms of vitamins and trace minerals to achieve the pre-determined diet inclusion rate.

2) Vitamin sources: VA, retinyl acetate; VD3, cholecalciferol; VE, D, L-α-tocopherol acetate; VK3, menadione sodium bisulfite; VB1, thiamine mononitrate; VB2, riboflavin; VB3, nicotinic acid; VB5, D-calcium pantothenate; VB6, pyridoxine hydrochloride; VB7, biotin; Vitamin B9, folic acid; Vitamin B12, cyanocobalamin; choline, choline chloride.

3) Trace mineral source: Cu, CuSO4; I, Ca(IO3)2; Fe, FeSO4; Mg, MnO; Se, NaSeO2; Zn, ZnO.

Table 2
Analyzed initial values of vitamin in vitamin and vitamin-trace mineral premixes
Item Vitamin premix 1 Vitamin premix 2 VTM premix 1 VTM premix 2 VTM premix 3 VTM premix 4
Vitamin1) (unit/kg)
 VA (IU) 5,317,420 5,350,883 1,367,333 1,393,358 1,358,165 1,382,106
 VD3 (IU) 1,169,405 1,197,127 300,297 302,709 301,525 303,454
 VE (IU) 12,044 11,996 2,942 2,942 2,963 2,979
 VK3 (mg) 1,171 1,164 297 306 294 293
 VB1 (mg) 1,212 1,179 303 302 299 307
 VB2 (mg) 2,436 2,487 585 597 589 601
 VB3 (mg) 12,167 12,015 3,017 3,019 2,984 3,018
 VB5 (mg) 7,173 7,226 1,836 1,785 1,768 1,847
 VB6 (mg) 1,202 1,191 302 296 298 306
 Choline (mg) - 166,713 - 44,333 - 43,448

Values represent means of six replicate samples each analyzed in duplicate.

VTM premix, vitamin-trace mineral premix; VA, vitamin A; VD3, vitamin D3; VE, vitamin E; VK3, vitamin K3; VB1, vitamin B1; VB2, vitamin B2; VB3; vitamin B3; VB5, vitamin B5; VB6, vitamin B6.

1) Vitamin premix 1 contained no choline chloride, Vitamin premix 2 contained 160,000 mg/kg of choline chloride. VTM premix 1 contained 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO, VTM premix 2 contained 40,000 mg/kg of choline chloride, 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO. VTM premix 3 contained 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO. VTM premix 4 contained 40,000 mg/kg of choline chloride, 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO.

Table 3
Probabilities of interactive and main effects of storage time and choline on stability (as defined by percentage of initial vitamin activity) of vitamins in vitamin premixes
Item Interactive effects Main effects


Time×choline Time Choline
VA 0.628 0.096 0.076
VD3 0.644 <0.001 0.958
VE 0.987 0.957 0.433
VK3 <0.001 <0.001 <0.001
VB1 0.962 <0.001 0.273
VB2 0.949 <0.001 0.742
VB3 0.934 0.493 0.111
VB5 0.999 <0.001 0.053
VB6 0.557 <0.001 0.898

VA, vitamin A; VD3, vitamin D3; VE, vitamin E; VK3, vitamin K3; VB1, vitamin B1; VB2, vitamin B2; VB3; vitamin B3; VB5, vitamin B5; VB6, vitamin B6.

Table 4
Probabilities of interactive and main effects of storage time, choline, and Cu and Zn elements on stability (as defined by percentage of initial vitamin activity) of vitamins in vitamin-trace mineral premixes
Item VA VD3 VE VK3 VB1 VB2 VB3 VB5 VB6
Interactive effects
 Time×choline×Cu/Zn 0.310 0.868 0.680 0.002 0.013 0.009 0.984 0.993 0.654
 Time×choline 0.011 0.969 0.899 <0.001 <0.001 <0.001 0.568 0.973 0.052
 Time×Cu/Zn <0.001 <0.001 0.320 <0.001 <0.001 0.001 0.729 0.993 <0.001
 Choline×Cu/Zn 0.075 0.316 0.925 <0.001 0.603 0.653 0.846 0.500 0.104
Main effects
 Time <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
 Choline <0.001 0.404 0.812 <0.001 <0.001 <0.001 0.886 0.483 0.848
 Cu/Zn <0.001 <0.001 0.970 <0.001 <0.001 <0.001 0.937 0.740 <0.001

VA, vitamin A; VD3, vitamin D3; VE, vitamin E; VK3, vitamin K3; VB1, vitamin B1; VB2, vitamin B2; VB3; vitamin B3; VB5, vitamin B5; VB6, vitamin B6.

Table 5
Prediction equations for vitamin retention (%) in different vitamin-trace mineral premixes during storage
No. Item Types1) Predicted equations2) R2 RMSEP p-value
1 VA VTM premix 1 y = −5.706x+106.014 0.998 1.158 <0.001
2 VA VTM premix 2 y = −8.967x+106.085 0.974 7.573 0.002
3 VA VTM premix 3 y = 144.432e−0.286x 0.989 0.151 <0.001
4 VA VTM premix 4 y = 159.533e−0.503x 0.882 0.944 0.018
5 VD3 VTM premix 1 y = 100.97e−0.025x 0.977 0.020 0.002
6 VD3 VTM premix 2 y = 102.152e−0.027x 0.985 0.017 0.001
7 VD3 VTM premix 3 y = 98.338e−0.041x 0.919 0.063 0.010
8 VD3 VTM premix 4 y = 95.57e−0.039x 0.900 0.066 0.014
9 VE VTM premix 1 y = 98.111e−0.008x 0.797 0.020 0.042
10 VE VTM premix 2 y = 98.505e−0.008x 0.944 0.010 0.006
11 VE VTM premix 3 y = 97.331e−0.006x 0.756 0.017 0.055
12 VE VTM premix 4 y = 97.288e−0.006x 0.554 0.026 0.146
13 VK3 VTM premix 1 y = 103.721e−0.058x 0.992 0.028 <0.001
14 VK3 VTM premix 2 y = 101.025e−0.068x 0.945 0.085 0.006
15 VK3 VTM premix 3 y = 108.391e−0.087x 0.989 0.047 0.001
16 VK3 VTM premix 4 y = 112.062e−0.152x 0.975 0.125 0.002
17 VB1 VTM premix 1 y = 102.21e−0.054x 0.970 0.042 0.001
18 VB1 VTM premix 2 y = 98.939e−0.094x 0.799 0.240 0.041
19 VB1 VTM premix 3 y = 99.533e−0.081x 0.876 0.157 0.019
20 VB1 VTM premix 4 y =133.472e−0.242x 0.992 0.095 <0.001
21 VB2 VTM premix 1 y = 97.178e−0.01x 0.810 0.026 0.037
22 VB2 VTM premix 2 y = 90.3e−0.012x 0.492 0.062 0.187
23 VB2 VTM premix 3 y = 97.393e−0.003x 0.477 0.018 0.217
24 VB2 VTM premix 4 y = 94.924e−0.015x 0.861 0.031 0.023
25 VB3 VTM premix 1 y = 98.349e−0.004x 0.838 0.009 0.029
26 VB3 VTM premix 2 y = 99.114e−0.005x 0.904 0.009 0.013
27 VB3 VTM premix 3 y = 99.292e−0.006x 0.774 0.016 0.049
28 VB3 VTM premix 4 y = 99.252e−0.006x 0.896 0.010 0.015
29 VB5 VTM premix 1 y = 100.996e−0.006x 0.884 0.012 0.017
30 VB5 VTM premix 2 y = −0.672x+101.276 0.887 1.231 0.017
31 VB5 VTM premix 3 y = −0.647x + 100.34 0.907 0.908 0.008
32 VB5 VTM premix 4 y = −0.62x + 101.288 0.913 0.840 0.007
33 VB6 VTM premix 1 y = −1.946x + 101.076 0.998 0.437 <0.001
34 VB6 VTM premix 2 y = 101.315e−0.018x 0.984 0.012 0.001
35 VB6 VTM premix 3 y = 101.667e−0.027x 0.810 0.067 0.037
36 VB6 VTM premix 4 y = 97.419e−0.02x 0.747 0.060 0.059

VTM, vitamin-trace mineral; RMSEP, root mean square error of prediction; VA, vitamin A; VD3, vitamin D3; VE, vitamin E; VK3, vitamin K3; VB1, vitamin B1; VB2, vitamin B2; VB3; vitamin B3; VB5, vitamin B5; VB6, vitamin B6.

1) VTM premix 1 contained 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO; VTM premix 2 contained 40,000 mg/kg of choline chloride, 500 mg/kg of CuSO4 and 8,000 mg/kg of ZnO; VTM premix 3 contained 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO; VTM premix 4 contained 40,000 mg/kg of choline chloride, 20,000 mg/kg of CuSO4 and 225,000 mg/kg of ZnO.

2) y (%) is retention of vitamin, x (month) is storage time.

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