The stability of vitamin C and other vitamins in the diets of breeding hens

Animal Feed Science and Technology 177 (2012) 253–258

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The stability of vitamin C and other vitamins in the diets of breeding hens M. Skˇrivan ∗ , M. Englmaierová, I. Bubancová, G. Dlouhá Institute of Animal Science, Pˇrátelství 815, Prague, CZ 104 00, Czech Republic

a r t i c l e

i n f o

Article history: Received 5 October 2011 Received in revised form 18 June 2012 Accepted 22 June 2012

Keywords: Vitamins Stability Mixed feed Pelleting

a b s t r a c t The stability of vitamin C in mash and pelleted mixed feed was studied during a 12-week period of guaranteed storage time. This research involved three feed types: feed without the addition of ascorbic acid (AA) and mixed feed supplemented with 200 mg/kg AA in the form of crystalline or protected AA (Monophosphate, Lutavit® C Monophosphate 35). The other vitamins were part of a vitamin–mineral premix. Vitamin C concentrations were analysed using 14-day intervals, with other vitamins being evaluated at the beginning and after 4 and 12 weeks of storage. The interaction between the source of vitamin C and the pelleting (P<0.001), between the source of vitamin C and the storage time (P<0.001) and the combined effect of the pelleting and the storage time (P<0.001) was ascertained in the vitamin C content in the feed mixture. Monophosphate was highly stable in the mixture until the 10th week of storage and seems to be an appropriate source of vitamin C in animal feed. In contrast, crystalline AA had notably low stability. The process of pelleting of the diet had a different effect on the stability of the monitored vitamins. During storage, the stability of vitamin C and retinol was lower and ␣-tocopherol and ␤-carotene stability was higher in the pelleted feed than in the non-pelleted feed. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Chickens may not have the ability to produce sufficient ascorbic acid (AA) to meet their physiological needs (Pardue and Thaxton, 1986). Vitamin C is an anti-stress factor and is therefore important for various types of poultry during periods of high temperatures. Ascorbic acid positively affects reproduction. Chickens require vitamin C for amino acid and mineral metabolism and for the synthesis of hormones, such as testosterone (McDowell, 1989). Thus, breeding poultry need ascorbic acid throughout the year. The effects of dietary vitamin C in poultry can vary widely because of the low stability of previously added sources of ascorbic acid. However, vitamin C is subject to oxidative and enzymatic degradation to dehydroascorbic acid and subsequently diketogulonic acid; the vitamin therefore loses its potency (Nyyssonen et al., 2000). Various factors, including light, metal ions, alkaline pH and high temperature, influence the content of vitamin C in feed. High temperatures during extrusion significantly reduced the vitamins in fish feed (Marchetti et al., 1999; Anderson and Sunderland, 2002). Additionally, the stability of coated vitamin C in aquaculture feed was reduced by 50%, due to 110–150 ◦ C temperatures during extrusion (Gadient and Fenster, 1994). In poultry, crystalline AA is still mostly added to the drinking water of breeding hens, which is quite impractical. The supplementation of pig feed with crystalline AA led to a decline of 31–44% of the initially

∗ Corresponding author. Tel.: +420 267009720; fax: +420 267710779. E-mail address: [email protected] (M. Skˇrivan). 0377-8401/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2012.06.010

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M. Skˇrivan et al. / Animal Feed Science and Technology 177 (2012) 253–258

Table 1 The composition of the control feed mixture.a Ingredient

g/kg

Wheat Maize Wheat bran Soybean meal Fish meal Rapeseed oil Dicalcium phosphate Sodium chloride Fine limestone dl-Methionine Vitamin–mineral premixb

560 190 10 130 12 10 15 2 65.8 0.2 5

Analysed nutrient contentc

g/kg

Dry matter Crude protein Fat Crude fibre Calcium Total phosphorus Non-phytate phosphorus Calculated MEN (MJ/kg)d

898 152 21 33 30.2 4.71 3.94 11.56

a Another experimental feed mixture was supplemented with 200 mg of vitamin C/kg in the form of crystalline ascorbic acid or ascorbic acid monophosphate. b The vitamin–mineral premix provided the following per kg of diet: retinylacetate 2.5 mg, cholecalciferol 60 ␮g, dl-␣-tocopheryl acetate 50 mg, niacin 20 mg, Ca pantothenate 6 mg, thiamine 1.5 mg, riboflavin 4 mg, pyridoxine 2 mg, folic acid 0.4 mg, biotin 0.06 mg, cobalamin 0.01 mg, choline Cl 250 mg, butylated hydroxytoluene 27 mg, Mn 60 mg, Zn 50 mg, Fe 30 mg, Cu 6 mg, I 0.7 mg, Co 0.3 mg, and Se 0.2 mg. c The data are expressed as fed basis. d Feed tables of Zelenka et al. (2007) were used for calculation of metabolisable energy (ME).

added amounts due to pelleting according to the above allowances of 150–600 mg/kg (Gebert et al., 2006). Today, several pharmaceutical companies offer stabilised vitamin C, which is designed for incorporation into animal feed. Independent tests of the stability of AA in stored diets during guaranteed storage have not been conducted. There are particularly scarce data available concerning pig feed regarding the change of concentration of supplemented AA in mixed feed immediately after preparation, rather than during storage (Eichenberger et al., 2004; Lahuˇcky´ et al., 2005; Gebert et al., 2006). Skˇrivan et al. (2012) reported on the stability of AA coated with ethyl cellulose in the feed of chickens after 5 weeks. The recommended storage time of mixed feed by manufacturers is up to 12 weeks. Therefore, the objective of this study was to investigate the stability of crystalline and stabilised AA in mash and pelleted feed for breeders over a period of 12 weeks. The stabilities of ␣-tocopherol, retinol and ␤-carotene were monitored simultaneously.

2. Materials and methods 2.1. Experimental design The experiment was conducted for mixed feed for meat-type hens in reproductive and breeding farms without vitamin C addition (control, Table 1) and with mixed feeds enriched with AA (200 mg/kg) in two different forms: crystalline AA (Crystalline, content at least 999 g/kg; Trouw Nutrition Biofaktory s.r.o., Prague, Czech Republic) and protected AA (Monophosphate, Lutavit® C Monophosphate 35; produced by Orffa Nederland Feed B.V., Giessen, the Netherlands; provided by Trouw Nutrition Biofaktory s.r.o., Prague, Czech Republic). The protected Lutavit® contains at least 350 g/kg of AA; the active ingredient is l-ascorbic acid-2-monophosphate-calcium sodium salt × 3H2 O; the molar mass is 319.1 g/mol; the bulk density is 0.6–0.8 g/cm3 ; and the particle size is 98% <0.5 mm. Ascorbic acid was supplied in a mixture as part of the vitamin–mineral premix Aminovitan NP-M (Trouw Nutrition Biofaktory s.r.o., Prague, Czech Republic). The time difference between preparing the vitamin–mineral premix and its mixing in hen feed was 5 days. The mixed feed was mixed by the International Poultry Testing of the Czech Republic (MTD, s.p., branch in Lysá nad Labem, Czech Republic). Two hundred kilogrammes of mixed feed was produced for each form of AA and control, of which 100 kg was not pelleted, and 100 kg was pelleted. The pelleting process began with 20 min of homogenisation. The homogenised mixture was steamed using a gas generator at a temperature of 60 ◦ C. The pressure generated by pushing mash through a pellet mill increased the temperature by 10 ◦ C. The pellets were at this temperature for only 1 min, after which time they were fan-cooled at the ambient temperature (max. of up to 5 ◦ C higher than the ambient temperature). The pellet mill die had holes that measured 3.0 mm in diameter.

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Table 2 The ascorbic acid content in the feed mixture (mg/kg) – estimated effects with standard error (S.E.) of estimate for each effect. SC × TS

Beginning

2nd week

4th week

6th week

8th week

10th week

12th week

S.E.

Control Crystalline Monophosphate

2.88 42.41 186.28

3.14 45.95 178.06

3.12 45.8 186.14

2.62 44.17 184.58

2.60 34.14 183.19

2.52 36.13 179.47

2.54 21.60 164.89

±2.47 ±2.47 ±2.47

P × TS

Beginning

2nd week

4th week

6th week

8th week

10th week

12th week

S.E.

91.32 65.38

89.34 64.9

82.19 64.43

Not pelleted Pelleted

86.59 67.76

83.9 67.53

84.26 61.15

±2.02 ±2.02

69.14 56.88

SC × P

Not pelleted

Pelleted

S.E.

Control Crystalline Monophosphate

2.76 51.87 196.83

2.79 25.33 163.91

±1.32 ±1.32 ±1.32

Predictor

SCa

Pa

TSa

SC × Pa

SC × TSa

P × TS

SC × P × TS

Probability

<0.001

<0.001

<0.001

<0.001

<0.001

<0.010

NS

SC, the source of vitamin C; P, the form of diet (the pelleting); TS, the storage time; NS, not significant; n = 6. a Stable effect.

2.2. Samples The mixed feed was stored in 100 kg quantities in paper bags (100 kg per treatment) in the dark at a temperature of 18–20 ◦ C, and the relative humidity was maintained between 50% and 60% in an air-conditioned room. The mixed feed samples that were used to determine the vitamin C content were analysed at the beginning of the experiment and after storage at 2, 4, 6, 8, 10, and 12 weeks. The content of ␣-tocopherol, retinol and ␤-carotene was ascertained at the beginning of the experiment and at the 4th and 12th weeks of storage. At each collection interval, the samples of mixed feed were obtained from bags by the sampler and processed using the quartering method. Each sample weighed approximately 50 g. Six samples from each group were collected in the term. 2.3. Laboratory analyses The samples were taken immediately after feed production (at the beginning of the experiment) and during other collection times and were analysed immediately. The ␣-tocopherol, retinol and ␤-carotene contents of the feed mixture were determined in accordance with the EN 12822, EN 12823-1 and EN 12823-2 European standards (2000) by a Shimadzu HPLC system (VP series; Shimadzu, Kyoto, Japan) equipped with a diode-array detector. The standards used were (±)-␣-tocopherol (purity ≥97.0%), retinol (purity ≥97.0%), and ␤-carotene (purity >97.0%; Fluka, Sigma–Aldrich, Steinheim, Germany). A modified EN 14130 European standard (2004) was used to determine the vitamin C (ascorbic acid and dehydroascorbic acid) content. The feed samples were homogenised, and vitamin C was extracted using 2% metaphosphoric acid (MPA). The samples were filtered through a 0.22-␮m membrane filter and were analysed by HPLC using the Synergi 4 ␮ Fusion-RP 80A column (Phenomenex, Torrance, CA, USA) and a gradient elution (25 mM KH2 PO4 :acetonitrile). The pH of the 25 mM KH2 PO4 was adjusted to 3 using 20% MPA. l (+)-Ascorbic acid puriss (purity ≥99.7%; Sigma–Aldrich, Steinheim, Germany) was used as a standard. 2.4. Statistical analysis Statistical analysis was performed by analysis of variance (ANOVA) using PROC MIXED (SAS v9.2). In each model, the fixed effects were the source of vitamin C (SC), the form of diet (the pelleting, P) and the storage time (TS), as well as their interactions of SC × P, SC × TS and P × TS. The interaction SC × P × TS was omitted from models because this effect was not significant. A 5% significance level was determined. Information about the stable effects of these models was recorded in Tables 2–5. The results for each vitamin are presented in tables with the estimated means for each interaction with respect to the other fixed effects in the model (statement LSMEANS in PROC MIXED, SAS v9.2). The last column in each table denotes the standard error of the estimated mean (S.E.), which is the same for each row in the particular table because of the balanced design of the experiment. 3. Results and discussion The concentration of vitamin C in the diet, monitored at 2-week intervals, is shown in Table 2. All three two-way interactions were ascertained in the vitamin C content in the feed mixture. The concentration of vitamin C remained stable

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Table 3 The ␣-tocopherol content in the feed mixture (mg/kg) – estimated effects with standard error (S.E.) of estimate for each effect. SC × TS

Beginning

4th week

12th week

S.E.

Control Crystalline Monophosphate

75.98 82.66 81.17

80.14 82.97 87.57

74.52 73.95 79.10

±2.21 ±2.21 ±2.21

P × TS

Beginning

4th week

12th week

S.E.

Not pelleted Pelleted

79.73 80.14

80.38 86.73

71.10 80.62

±1.81 ±1.81

SC × P

Not pelleted

Pelleted

S.E.

Control Crystalline Monophosphate

74.77 75.93 80.51

78.99 83.78 84.72

±1.81 ±1.81 ±1.81

Predictor

SCa

Pa

TSa

SC × Pa

SC × TS

P × TSa

SC × P × TS

Probability

<0.010

<0.001

<0.001

NS

NS

<0.050

NS

SC, the source of vitamin C; P, the form of diet (the pelleting); TS, the storage time; NS, not significant; n = 6. a Stable effect. Table 4 The retinol content in the feed mixture (mg/kg) – estimated effects with standard error (S.E.) of estimate for each effect. SC × TS

Beginning

4th week

12th week

S.E.

Control Crystalline Monophosphate

4.15 3.66 4.84

2.86 3.19 4.85

2.66 2.47 3.64

±0.31 ±0.31 ±0.31

P × TS

Beginning

4th week

12th week

S.E.

Not pelleted Pelleted

5.21 3.22

3.92 3.34

2.91 2.93

±0.25 ±0.25

SC × P

Not pelleted

Pelleted

S.E.

Control Crystalline Monophosphate

3.38 3.37 5.29

3.06 2.85 3.59

±0.25 ±0.25 ±0.25

Predictor

SCa

Pa

TSa

SC × P

SC × TSa

P × TSa

SC × P × TS

Probability

<0.001

<0.001

<0.001

<0.050

NS

<0.001

NS

SC, the source of vitamin C; P, the form of diet (the pelleting); TS, the storage time; NS, not significant; n = 6. a Stable effect. Table 5 The ␤-carotene content in the feed mixture (mg/kg) – estimated effects with standard error (S.E.) of estimate for each effect. SC × TS

Beginning

4th week

12th week

S.E.

Control Crystalline Monophosphate

0.140 0.113 0.126

0.122 0.117 0.123

0.086 0.087 0.084

±0.004 ±0.004 ±0.004

P × TS

Beginning

4th week

12th week

S.E.

Not pelleted Pelleted

0.130 0.123

0.110 0.132

0.075 0.096

±0.003 ±0.003

SC × P

Not pelleted

Pelleted

S.E.

Control Crystalline Monophosphate

0.111 0.100 0.104

0.121 0.111 0.118

±0.003 ±0.003 ±0.003

Predictor

SC

Pa

TSa

SC × Pa

SC × TS

P × TSa

SC × P × TS

Probability

<0.010

<0.001

<0.001

NS

<0.050

<0.001

NS

SC, the source of vitamin C; P, the form of diet (the pelleting); TS, the storage time; NS, not significant; n = 6. a Stable effect.

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only in the mixture supplemented with monophosphate until the 10th week of storage (SC × TS; P<0.001). Crystalline AA underwent the greatest decline (by 79%) between the preparation of the vitamin–mineral premix enriched by AA and its mixing into the hen diet, when crystalline AA came into contact with heavy metals from the premix. l-ascorbic acid is unstable when exposed to either oxygen or certain minerals, and this instability can result in rapid oxidation to the dehydroascorbic acid derivative. The results show that it is completely inappropriate to add crystalline AA into the diet. Vitamin C decreased in the feed supplemented with protected monophosphate was by 8% at the beginning of the experiment and by 19% after 12 weeks of storage. Conversely, Lahuˇcky´ et al. (2005) showed a high loss of vitamin C (35%) in freshly mixed pig feed enriched with a protected source of vitamin C in combination with ␣-tocopherol. A relatively low stability (though higher than crystalline AA) was observed in an experiment with another type of stabilised vitamin C, ethyl cellulose coated AA, ROVIMIX® C-EC (Skˇrivan et al., 2012). A higher addition (560 mg/kg) of coated AA decreased after 35 days to 78–68% and a lower addition (280 mg/kg) decreased to 45–36% of the initial amount. Thus, higher supplementation meant greater protection against oxidation. The negative effect of pelleting on the stability of vitamin C was found during storage (P × TS; P<0.010) and in the source of vitamin C (SC × P; P<0.001). The highest values of vitamin C content were determined in non-pelleted diets stored until the 10th week and in the non-pelleted diet supplemented with monophosphate. The reduction of crystalline AA during feed pelleting and subsequent storage was shown in Gebert et al. (2006). After the addition of 150, 300 and 600 mg/kg of AA, the vitamin C content declined to only 31%, 37% and 44% of the initial added amount of AA. This finding is in accordance with Eichenberger et al. (2004). In addition, Creel et al. (2001) showed that feed stored under dry heat resulted in a linear decrease in the AA content, and the rate of decline was 5-fold higher in the supplemented diet. The significant interaction between the form of diet and the storage time was ascertained in the concentration of ␣tocopherol content (P × TS; P<0.05; Table 3). The highest value was found in the 4-week stored and pelleted feed mixture in comparison with the non-pelleted diet at the 12th week of storage. The concentration of ␣-tocopherol in the mash and pelleted feed immediately after preparation was not significantly different. Gebert et al. (2006) also did not find a decrease of ␣-tocopherol after pelleting. The addition of AA significantly increased the ␣-tocopherol content in the feed mixture. ␣-Tocopherol content probably increased due to the antioxidant characteristics of ascorbic acid provided both in crystalline form and as monophosphate during the first 5 days, i.e., the time between preparing the vitamin–mineral premix and its mixing into the hen diet. Thus, protected ascorbic acid acts as an antioxidant, because the protection of AA against degradation is not complete. It is known that vitamin E works together with other cellular antioxidants, such as with the ascorbate system. During encounters with free radicals, vitamin E becomes deficient in electrons, which is compensated for by ascorbic acid. Thus, vitamin E molecules are available again to function as antioxidants. This result confirms the data on the synergy of AA and ␣-tocopherol as antioxidants (Bender, 2003). The stability of ␣-tocopherol is possible to a certain degree compared with the data from Tavˇcar-Kalcher and Vanguˇst (2007), who observed the stability of lipophilic vitamins in premixes without minerals. After 3 months, the content of ␣-tocopherol in their study declined by 6% and by 2–11% in the diets presented here. Pelleting in combination with storage time (P × TS; P<0.001) and in combination with vitamin C source (SC × P; P<0.050) decreased the retinol content in the feed (Table 4). The decrease in the retinol concentration due to pelleting was 38%. Jones (1986) found only an approximately 6.5% lower content of retinol after the pelleting of poultry feed. Different compositions of mixed feed, including a premix of vitamins and trace elements, could produce different results. When comparing the stability of vitamin C versus the stability of retinol, a significant interaction between retinol and vitamin C source (P<0.001; stable effect) was found. This means that a different degree of vitamin C stability for each type of vitamin C source is dependent on the retinol content. The combined effect of storage time with vitamin C source (SC × TS; P<0.050) and with the pelleting (P × TS; P<0.001) was observed in the ␤-carotene content (Table 5). A significantly higher ␤-carotene stability was found in pelleted feed mixtures compared to non-pelleted diets during storage. Information about the stability of ␤-carotene in the mixed feed is not available in the literature. Shofian et al. (2011) evaluated the effect of freeze-drying on antioxidant compounds of fruits and determined that freeze-drying did not exert any considerable effect on ␤-carotene concentration of fruits. The stability of ␤-carotene towards oxidation depends on its dispersion form and can be increased through encapsulation (Cao-Hoang et al., 2011). The pelleting of diet had a different effect on the stability of the evaluated vitamins. During the storage of mixed feed, the stability of vitamin C and retinol decreased and ␣-tocopherol and ␤-carotene stability increased by pelleting. We found no comparisons in the literature about the positive impact of pelleting on the stability of ␣-tocopherol or ␤-carotene in stored mixed feed.

4. Conclusions Protected AA in form of monophosphate (Lutavit® C Monophosphate 35) showed a high stability in mash and even in pelleted mixed feed during a guaranteed period of 12 weeks. In contrast, crystalline AA was so unstable that it was not suitable for incorporation into the complete mixed feed. Pelleting increased the stability of ␣-tocopherol and ␤carotene in the diet with the tested compositions. The dietary concentration of vitamin C and retinol decreased, due to pelleting.

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